scholarly journals Gene-Trait Matching and Prevalence of Nisin Tolerance Systems in Lactococus lactis

2021 ◽  
Vol 9 ◽  
Author(s):  
Lieke A. van Gijtenbeek ◽  
Thomas H. Eckhardt ◽  
Lucía Herrera-Domínguez ◽  
Elke Brockmann ◽  
Kristian Jensen ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Specific Effect ◽  
Starter Cultures ◽  
Main Mechanism ◽  
Effective Agent ◽  
Gram Positive ◽  
Gram Positive Bacteria

Lactococcus lactis cheese starter cultures typically contain a mix of many strains and may include variants that produce and/or tolerate the antimicrobial bacteriocin nisin. Nisin is well-established as an effective agent against several undesirable Gram-positive bacteria in cheese and various other foods. In the current study, we have examined the effect of nisin on 710 individual L. lactis strains during milk fermentations. Changes in milk acidification profiles with and without nisin exposure, ranging from unaltered acidification to loss of acidification, could be largely explained by the type(s) and variants of nisin immunity and nisin degradation genes present, but surprisingly, also by genotypic lineage (L. lactis ssp. cremoris vs. ssp. lactis). Importantly, we identify that nisin degradation by NSR is frequent among L. lactis and therefore likely the main mechanism by which dairy-associated L. lactis strains tolerate nisin. Insights from this study on the strain-specific effect of nisin tolerance and degradation during milk acidification is expected to aid in the design of nisin-compatible cheese starter cultures.

Lysozyme Treatment Makes 16S rRNA Amplicon Sequencing Results Less Biased

2021 ◽  
Author(s):  
Yuting Jiao ◽  
Zijie Gao ◽  
Shiyu Gui ◽  
Lu Ren ◽  
Yongyue Lu ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Community Analysis ◽  
Amplicon Sequencing ◽  
Microbial Community Analysis ◽  
Effective Agent ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
16S Rrna Amplicon Sequencing ◽  
Structure Result ◽  
Total Bacteria

Abstract Background Amplicon sequencing is widely applied in gut bacteria structure analysis. However, the proportion of Gram-positive bacteria may greatly affect the results of microbial community analysis. Lysozyme is an effective agent to extract DNA of Gram-positive bacteria. In this study, we assessed the influence of lysozyme treatment on results of Bactrocere dorsalis rectal bacteria structure. Result The results indicated that the total bacteria content can be significantly increased in lysozyme treated samples. Moreover, rectal bacteria diversity was significantly higher in lysozyme treated samples. A detail analysis revealed that abundance of Gram-positive bacteria significantly increased in samples treated with lysozyme. Conclusion This study indicates that lysozyme treatment before DNA extraction is an effective way to reduce bias in bacteria structure analysis, especially for samples with high proportion of Gram-positive bacteria.

scholarly journals Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Irina Sadovskaya ◽  
Evgeny Vinogradov ◽  
Pascal Courtin ◽  
Julija Armalyte ◽  
Mickael Meyrand ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lactococcus Lactis ◽  
Abc Transporter ◽  
Present Report ◽  
Gram Positive ◽  
Narrow Host Range ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
Major Structural Component ◽  
Dependent Pathway

ABSTRACT Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan. IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus Lactococcus lactis, a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the L. lactis cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci. IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus Lactococcus lactis, a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the L. lactis cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci.

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 An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

1998 ◽  
Vol 180 (16) ◽  
pp. 4324-4324
Author(s):  
Isabelle Poquet ◽  
S. Dusko Ehrlich ◽  
Alexandra Gruss
Keyword(s):  
Lactococcus Lactis ◽  
Gram Positive ◽  
Gram Positive Bacteria

Lactococcus lactis TrxD represents a subgroup of thioredoxins prevalent in Gram-positive bacteria containing WCXDC active site motifs

2014 ◽  
Vol 564 ◽  
pp. 164-172 ◽  
Author(s):  
Olof Björnberg ◽  
Petr Efler ◽  
Epie Denis Ebong ◽  
Birte Svensson ◽  
Per Hägglund
Keyword(s):  
Lactococcus Lactis ◽  
Active Site ◽  
Gram Positive ◽  
Gram Positive Bacteria

scholarly journals Synthesis, Characterization, and Antimicrobial Studies of Novel Series of 2,4-Bis(hydrazino)-6-substituted-1,3,5-triazine and Their Schiff Base Derivatives

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Hessa H. Al-Rasheed ◽  
Essam N. Sholkamy ◽  
Monirah Al Alshaikh ◽  
Mohammed R. H. Siddiqui ◽  
Ahmed S. Al-Obaidi ◽  
...  
Keyword(s):  
Schiff Base ◽  
Pathogenic Bacteria ◽  
Specific Effect ◽  
Antagonistic Effect ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Antimicrobial Studies ◽  
Schiff Base Derivatives ◽  
Wide Range

The present work represents the synthesis, characterization, and antimicrobial studies of novel series of 2,4-bis(hydrazino)-6-substituted-1,3,5-triazine and their Schiff base derivatives. IR, NMR (H1 and C13), elemental analysis, and LC-MS characterized the prepared compounds. The biological activity of the target products was evaluated as well. Twenty-two of the prepared compounds were selected according to their solubility in aqueous DMSO. Only eight compounds showed good activity against the selected pathogenic bacteria and did not show antagonistic effect against fungus Candida albicans. Two compounds 4k and 5g have wide-range effect presently in Gram-positive and Gram-negative bacteria while other compounds (4f, 4i, 4m, 5d, 6i, and 6h) showed specific effect against the Gram-negative or Gram-positive bacteria. The minimum inhibitory concentration (MIC, μg/mL) of 4f, 4i, 4k, and 6h compounds against Streptococcus mutans was 62.5 μg/mL, 100 μg/mL, 31.25 μg/mL, and 31.25 μg/mL, respectively. The MIC of 4m, 4k, 5d, 5g, and 6h compounds against Staphylococcus aureus was 62.5 μg/mL, 31.25 μg/mL, 31.25 μg/mL, 100 μg/mL, and 62.5 μg/mL, respectively. The MIC of 4k, 5g, and 6i compounds against Salmonella typhimurium was 31.25 μg/mL, 100 μg/mL, and 62.5 μg/mL, respectively. The MIC of 6i compound against Escherichia coli was 62.5 μg/mL.

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

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.

scholarly journals Disruption and Analysis of the clpB,clpC, and clpE Genes in Lactococcus lactis: ClpE, a New Clp Family in Gram-Positive Bacteria

1999 ◽  
Vol 181 (7) ◽  
pp. 2075-2083 ◽  
Author(s):  
Hanne Ingmer ◽  
Finn K. Vogensen ◽  
Karin Hammer ◽  
Mogens Kilstrup
Keyword(s):  
Heat Shock ◽  
Lactococcus Lactis ◽  
Wild Type ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Binding Domains ◽  
Folded Proteins ◽  
Zinc Binding Domain ◽  
Mutant Cells

ABSTRACT In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC,clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded byclpB was strongly induced by heat shock and, whileclpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L. lactisheat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis, could be necessary for degrading proteins generated by certain types of stress.

scholarly journals An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

1998 ◽  
Vol 180 (7) ◽  
pp. 1904-1912 ◽  
Author(s):  
Isabelle Poquet ◽  
S. Dusko Ehrlich ◽  
Alexandra Gruss
Keyword(s):  
Lactococcus Lactis ◽  
Genomic Library ◽  
Shuttle Vector ◽  
Nuclease Activity ◽  
Protein Export ◽  
Cell Fractionation ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Membrane Protein Topology ◽  
Export Signal

ABSTRACT The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of theStaphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called ΔSPNuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of ΔSPNuc to report protein export. The shuttle vector pFUN was designed to construct ΔSPNuc translational fusions whose expression signals are provided by inserted DNA. The capacity of ΔSPNuc to reveal and identify exported proteins was tested by generating anL. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All ΔSPNuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that ΔSPNuc is well suited to report both protein export and membrane protein topology.

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