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

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.

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Impact of Nisin-Producing Strains of Lactococcus lactis on the Contents of Bioactive Dipeptides, Free Amino Acids, and Biogenic Amines in Dutch-Type Cheese Models

Materials ◽

10.3390/ma13081835 ◽

2020 ◽

Vol 13 (8) ◽

pp. 1835 ◽

Cited By ~ 1

Author(s):

Monika Garbowska ◽

Antoni Pluta ◽

Anna Berthold-Pluta

Keyword(s):

Amino Acids ◽

Biogenic Amines ◽

Lactococcus Lactis ◽

Free Amino Acids ◽

Free Amino ◽

Total Content ◽

Microbial Activities ◽

Ripening Period ◽

Cheese Model

The goal of this study was to determine changes in contents of free amino acids, biogenic amines, and bioactive dipeptides (anserine and L-carnosine) in cheese models produced with the addition of nisin-producing strains of Lactococcus lactis over their ripening period. After 5 weeks of ripening, contents of total biogenic amines in the cheese models with the addition of L. lactis strains were lower than in the control cheese model. The cheese models examined differed significantly in contents of free amino acids through the ripening period. Individual free amino acids, such as ornithine, were found in some of the cheese models, which is indicative of their specific microbial activities. Both anserine and L-carnosine were detected in all variants of the cheese models. After 5-week ripening, the highest total content of bioactive dipeptides was determined in the cheese models produced with the nisin-producing culture of L. lactis 11454 (142.15 mg∙kg−1).

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Protease-Mediated Growth ofStaphylococcus aureuson Host Proteins Isopp3Dependent

mBio ◽

10.1128/mbio.02553-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 7

Author(s):

McKenzie K. Lehman ◽

Austin S. Nuxoll ◽

Kelsey J. Yamada ◽

Tammy Kielian ◽

Steven D. Carson ◽

...

Keyword(s):

Amino Acids ◽

Staphylococcus Aureus ◽

Glutamate Dehydrogenase ◽

Free Amino Acids ◽

Free Amino ◽

Nitrogen Sources ◽

Host Protein ◽

Dependent Manner ◽

Content Type ◽

Organ Systems

ABSTRACTStaphylococcus aureushas the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (ΔpckA) and glutamate dehydrogenase (ΔgudB), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important forS. aureusgrowth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can supportS. aureusgrowth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting thatS. aureushas the ability to detect peptides in the environment.IMPORTANCEStaphylococcus aureushas the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients forS. aureus. Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin supportS. aureusgrowth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by whichS. aureussenses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.

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Utilization of 15N-labelled yeast hydrolysate in Lactococcus lactis IL1403 culture indicates co-consumption of peptide-bound and free amino acids with simultaneous efflux of free amino acids

Antonie van Leeuwenhoek ◽

10.1007/s10482-013-0103-2 ◽

2014 ◽

Vol 105 (3) ◽

pp. 511-522 ◽

Cited By ~ 3

Author(s):

Kaspar Kevvai ◽

Mary-Liis Kütt ◽

Ildar Nisamedtinov ◽

Toomas Paalme

Keyword(s):

Amino Acids ◽

Lactococcus Lactis ◽

Free Amino Acids ◽

Free Amino ◽

Yeast Hydrolysate ◽

Lactis Il1403

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A Novel Mobilizing Tool Based on the Conjugative Transfer System of the IncM Plasmid pCTX-M3

Applied and Environmental Microbiology ◽

10.1128/aem.01205-20 ◽

2020 ◽

Vol 86 (17) ◽

Author(s):

Michał Dmowski ◽

Izabela Kern-Zdanowicz

Keyword(s):

Bacillus Subtilis ◽

Lactococcus Lactis ◽

Conjugative Transfer ◽

Conjugation System ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Gram Positive Bacteria ◽

Mobilizable Plasmid

ABSTRACT Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriTpCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria. We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis. Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells. IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, orf35 and orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis. The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.

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Lowering hydrophobic peptides and increasing free amino acids in cheese made with a Lactococcus lactis strain expressing a mutant oligopeptide binding protein

International Dairy Journal ◽

10.1016/j.idairyj.2005.09.021 ◽

2007 ◽

Vol 17 (3) ◽

pp. 218-225 ◽

Cited By ~ 4

Author(s):

A. Picon ◽

P. Gaya ◽

M. Nuñez

Keyword(s):

Amino Acids ◽

Lactococcus Lactis ◽

Free Amino Acids ◽

Free Amino ◽

Binding Protein ◽

Hydrophobic Peptides ◽

Lactis Strain

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Lysates of Metschnikowia yeast with higher content of hydroxyproline

BioResources ◽

10.15376/biores.15.2.3228-3236 ◽

2020 ◽

Vol 15 (2) ◽

pp. 3228-3236

Author(s):

Ewelina Pawlikowska ◽

Monika Szymańska ◽

Joanna Berłowska ◽

Dorota Kręgiel

Keyword(s):

Amino Acids ◽

Free Amino Acids ◽

Free Amino ◽

Amino Nitrogen ◽

Cell Lysis ◽

Chemical Characteristics ◽

Industrial Strains ◽

Chemical Profiles

The chemical characteristics of lysates obtained from yeasts belonging to Metschnikowia spp. were determined. Cell lysis was induced using saponin from Q. saponaria or 5% NaCl. The process was conducted at 50 °C for 24 for 48 h. The enzymatic profiles of the resulting lysates were analyzed. The mannose and glucose contents were also investigated, as well as the concentrations of proteins, free amino nitrogen (FAN), and free amino acids. The results were compared to the characteristics of lysates from conventional industrial strains of Saccharomyces spp. obtained under analogous conditions. The Metschnikowia lysates showed different chemical profiles and the pool of individual amino acids was generally smaller. However, the content of hydroxyproline HPro was 4 to 5 times higher. The results of this study show that yeast lysates are an attractive supplement for numerous applications.

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Changes in the concentrations of free amino acids in milk during growth of Lactococcus lactis indicate biphasic nitrogen metabolism

Journal of Dairy Research ◽

10.1017/s002202999700263x ◽

1998 ◽

Vol 65 (1) ◽

pp. 101-107 ◽

Cited By ~ 23

Author(s):

GORDON W. NIVEN ◽

DOROTHY J. KNIGHT ◽

FRANCIS MULHOLLAND

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Lactococcus Lactis ◽

Free Amino Acids ◽

Free Amino ◽

Total Concentration ◽

Milk Proteins ◽

Arginine Deiminase ◽

Logarithmic Phase ◽

Second Phase

Analysis of the concentrations of free amino acids in milk during growth of Lactococcus lactis subsp. lactis revealed a biphasic pattern of change during the logarithmic phase. During the first period there was little overall change in the total concentration of amino acids in the medium. The second phase was characterized by increased net liberation of free amino acids. There were also qualitative differences in the amino acids that were taken up and utilized during each period. The concentrations of Val, Leu and Ile decreased only during the early phase, while those of Ser, Arg, Thr and Met decreased only during the second phase. Gly and Ala were utilized throughout logarithmic growth. Gly uptake appeared to be greater during the second period and accounted for the largest proportion of free amino acid utilization at this time. It is possible that the biphasic nature of amino acid nutrition was due to increased consumption in late log phase of peptides derived from milk proteins by proteolysis. Increased activity of the arginine deiminase pathway during late log phase was inferred from increased utilization of Arg and liberation of citrulline and ornithine.

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