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.

Promoting acid resistance and nisin yield of Lactococcus lactis F44 by genetically increasing D-Asp amidation level inside cell wall

2017 ◽  
Vol 101 (15) ◽  
pp. 6137-6153 ◽  
Author(s):  
Panlong Hao ◽  
Dongmei Liang ◽  
Lijie Cao ◽  
Bin Qiao ◽  
Hao Wu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lactococcus Lactis ◽  
Acid Resistance ◽  
Nisin Yield

scholarly journals Lysis of a Lactococcus lactis Dipeptidase Mutant and Rescue by Mutation in the Pleiotropic Regulator CodY

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Chenxi Huang ◽  
Jhonatan A. Hernandez-Valdes ◽  
Oscar P. Kuipers ◽  
Jan Kok
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Nitrogen Metabolism ◽  
Lactococcus Lactis ◽  
Cell Shape ◽  
Single Point Mutation ◽  
Binding Motif ◽  
Acid Uptake ◽  
Cell Wall Synthesis ◽  
Content Type

ABSTRACT Lactococcus lactis subsp. cremoris MG1363 is a model for the lactic acid bacteria (LAB) used in the dairy industry. The proteolytic system, consisting of a proteinase, several peptide and amino acid uptake systems, and a host of intracellular peptidases, plays a vital role in nitrogen metabolism and is of eminent importance for flavor formation in dairy products. The dipeptidase PepV functions in the last stages of proteolysis. A link between nitrogen metabolism and peptidoglycan (PG) biosynthesis was underlined by the finding that deletion of the dipeptidase gene pepV (creating strain MGΔpepV) resulted in a prolonged lag phase when the mutant strain was grown with a high concentration of glycine. In addition, most MGΔpepV cells lyse and have serious defects in their shape. This phenotype is due to a shortage of alanine, since adding alanine can rescue the growth and shape defects. Strain MGΔpepV is more resistant to vancomycin, an antibiotic targeting peptidoglycan d-Ala–d-Ala ends, which confirmed that MGΔpepV has an abnormal PG composition. A mutant of MGΔpepV was obtained in which growth inhibition and cell shape defects were alleviated. Genome sequencing showed that this mutant has a single point mutation in the codY gene, resulting in an arginine residue at position 218 in the DNA-binding motif of CodY being replaced by a cysteine residue. Thus, this strain was named MGΔpepVcodYR218C. Transcriptome sequencing (RNA-seq) data revealed a dramatic derepression in peptide uptake and amino acid utilization in MGΔpepVcodYR218C. A model of the connections among PepV activity, CodY regulation, and PG synthesis of L. lactis is proposed. IMPORTANCE Precise control of peptidoglycan synthesis is essential in Gram-positive bacteria for maintaining cell shape and integrity as well as resisting stresses. Although neither the dipeptidase PepV nor alanine is essential for L. lactis MG1363, adequate availability of either ensures proper cell wall synthesis. We broaden the knowledge about the dipeptidase PepV, which acts as a linker between nitrogen metabolism and cell wall synthesis in L. lactis.

scholarly journals Nuclear Localization of Haa1, Which Is Linked to Its Phosphorylation Status, Mediates Lactic Acid Tolerance in Saccharomyces cerevisiae

2014 ◽  
Vol 80 (11) ◽  
pp. 3488-3495 ◽  
Author(s):  
Minetaka Sugiyama ◽  
Shin-Pei Akase ◽  
Ryota Nakanishi ◽  
Hitoshi Horie ◽  
Yoshinobu Kaneko ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Subcellular Localization ◽  
Target Genes ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Acid Resistance ◽  
Nuclear Accumulation ◽  
Content Type ◽  
Adaptation Response

ABSTRACTImprovement of the lactic acid resistance of the yeastSaccharomyces cerevisiaeis important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened forS. cerevisiaegenes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genesYGP1,GPG1, andSPI1, while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin geneMSN5led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

scholarly journals Engineering Trehalose Synthesis in Lactococcus lactis for Improved Stress Tolerance

2011 ◽  
Vol 77 (12) ◽  
pp. 4189-4199 ◽  
Author(s):  
Ana Lúcia Carvalho ◽  
Filipa S. Cardoso ◽  
Andreas Bohn ◽  
Ana Rute Neves ◽  
Helena Santos
Keyword(s):  
Lactococcus Lactis ◽  
Oral Drug Delivery ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Ph Control ◽  
Oral Drug ◽  
Resting Cells ◽  
Content Type ◽  
Food Grade ◽  
Cell Defense

ABSTRACTTrehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose inPropionibacterium freudenreichiiin response to acid stress, a result that led to the idea that endowingLactococcus lactiswith the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway ofL. lactis, i.e.,trePPandpgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given thatL. lactislacks trehalose 6-phosphate phosphatase, the respective gene,otsB, from the food-grade organismP. freudenreichiiwas used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.

scholarly journals Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

2011 ◽  
Vol 77 (10) ◽  
pp. 3327-3334 ◽  
Author(s):  
Thi Mai Huong To ◽  
Cosette Grandvalet ◽  
Raphaëlle Tourdot-Maréchal
Keyword(s):  
Fatty Acids ◽  
Stationary Phase ◽  
Lactococcus Lactis ◽  
Unsaturated Fatty Acids ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Phase Cell ◽  
Wild Type ◽  
Membrane Phospholipids ◽  
Content Type

ABSTRACTCyclopropane fatty acids (CFAs) are synthetizedin situby the transfer of a methylene group fromS-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacteriumLactococcus lactisMG1363. A mutant of thecfagene was constructed by allelic exchange. Thecfagene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of thecfagene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential forL. lactissubsp.cremorissurvival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

scholarly journals Contribution of YthA, a PspC Family Transcriptional Regulator ofLactococcus lactisF44 Acid Tolerance and Nisin Yield: a Transcriptomic Approach

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Hao Wu ◽  
Jingui Liu ◽  
Sen Miao ◽  
Yue Zhao ◽  
Hongji Zhu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Survival Rate ◽  
Transcriptome Analysis ◽  
Acid Stress ◽  
Acid Resistance ◽  
Transcriptional Regulator ◽  
Regulation Mechanism ◽  
Content Type ◽  
Regulation Mechanisms ◽  
Nisin Yield

ABSTRACTTo overcome the adverse impacts of environmental stresses during growth, different adaptive regulation mechanisms can be activated inLactococcus lactis. In this study, the transcription levels of eight transcriptional regulators ofL. lactissubsp.lactisF44 under acid stress were analyzed using quantitative reverse transcription-PCR. Eight gene-overexpressing strains were then constructed to examine their influences on acid-resistant capability. OverexpressingythA, a PspC family transcriptional regulator, increased the survival rate by 3.2-fold compared to the control at the lethal pH 3.0 acid shock. Moreover, the nisin yield was increased by 45.50%. TheythA-overexpressing strain FythA appeared to have higher intracellular pH stability and nisin-resistant ability. Subsequently, transcriptome analysis revealed that the vast majority of genes associated with amino acid biosynthesis, including arginine, serine, phenylalanine, and tyrosine, were predominantly upregulated in FythA. Arginine biosynthesis (argGandargH), arginine deiminase pathway, and polar amino acid transport (ysfEandysfF) were proposed to be the main regulation mechanisms of YthA. Furthermore, the transcription of genes associated with pyrimidine and exopolysaccharide biosynthesis were upregulated. The transcriptional levels ofnisIPRKFEGgenes were substantially higher in FythA, which directly contributed to the yield and resistance of nisin. Three potential DNA-binding sequences were predicted by computer analysis using the upstream regions of genes with prominent changes. This study showed that YthA could increase acid resistance and nisin yield and revealed a putative regulation mechanism of YthA.IMPORTANCENisin, produced byLactococcus lactissubsp.lactis, is widely used as a safe food preservative. Acid stress becomes the primary restrictive factor of cell growth and nisin yield. In this research, we found that the transcriptional regulator YthA was conducive to enhancing the acid resistance ofL. lactisF44. OverexpressingythAcould significantly improve the survival rate and nisin yield. The stability of intracellular pH and nisin resistance were also increased. Transcriptome analysis showed that nisin immunity and the biosynthesis of some amino acids, pyrimidine, and exopolysaccharides were enhanced in the engineered strain. This study elucidates the regulation mechanism of YthA and provides a novel strategy for constructing robust industrialL. lactisstrains.

scholarly journals Interplay between Antibiotic Efficacy and Drug-Induced Lysis Underlies Enhanced Biofilm Formation at Subinhibitory Drug Concentrations

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Wen Yu ◽  
Kelsey M. Hallinen ◽  
Kevin B. Wood
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Cell Lysis ◽  
Living Cells ◽  
Cell Wall Synthesis ◽  
Drug Induced ◽  
Trade Off ◽  
Content Type ◽  
Subinhibitory Concentrations ◽  
Antibiotic Efficacy

ABSTRACTSubinhibitory concentrations of antibiotics have been shown to enhance biofilm formation in multiple bacterial species. While antibiotic exposure has been associated with modulated expression of many biofilm-related genes, the mechanisms of drug-induced biofilm formation remain a focus of ongoing research efforts and may vary significantly across species. In this work, we investigate antibiotic-induced biofilm formation inEnterococcus faecalis, a leading cause of nosocomial infections. We show that biofilm formation is enhanced by subinhibitory concentrations of cell wall synthesis inhibitors but not by inhibitors of protein, DNA, folic acid, or RNA synthesis. Furthermore, enhanced biofilm is associated with increased cell lysis, increases in extracellular DNA (eDNA) levels, and increases in the density of living cells in the biofilm. In addition, we observe similar enhancement of biofilm formation when cells are treated with nonantibiotic surfactants that induce cell lysis. These findings suggest that antibiotic-induced biofilm formation is governed by a trade-off between drug toxicity and the beneficial effects of cell lysis. To understand this trade-off, we developed a simple mathematical model that predicts changes in antibiotic-induced biofilm formation due to external perturbations, and we verified these predictions experimentally. Specifically, we demonstrate that perturbations that reduce eDNA (DNase treatment) or decrease the number of living cells in the planktonic phase (a second antibiotic) decrease biofilm induction, while chemical inhibitors of cell lysis increase relative biofilm induction and shift the peak to higher antibiotic concentrations. Overall, our results offer experimental evidence linking cell wall synthesis inhibitors, cell lysis, increased eDNA levels, and biofilm formation inE. faecaliswhile also providing a predictive quantitative model that sheds light on the interplay between cell lysis and antibiotic efficacy in developing biofilms.

scholarly journals Agrobacterium tumefaciens Growth Pole Ring Protein: C Terminus and Internal Apolipoprotein Homologous Domains Are Essential for Function and Subcellular Localization

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
John Zupan ◽  
Zisheng Guo ◽  
Trevor Biddle ◽  
Patricia Zambryski
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Agrobacterium Tumefaciens ◽  
Polar Growth ◽  
Content Type ◽  
Growth Pole ◽  
Human Apolipoprotein ◽  
Apolipoprotein A ◽  
C Terminus ◽  
Organizing Center

ABSTRACT The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth. IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode—polar growth—is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

scholarly journals Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

2020 ◽  
Vol 203 (2) ◽  
pp. e00463-20
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Gene Product ◽  
Fluorescent Protein ◽  
Lytic Bacteriophage ◽  
Cell Wall Synthesis ◽  
Content Type ◽  
Ftsz Ring ◽  
Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

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