scholarly journals Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

2007 ◽  
Vol 73 (23) ◽  
pp. 7542-7547 ◽  
Author(s):  
Dag Anders Brede ◽  
Sheba Lothe ◽  
Zhian Salehian ◽  
Therese Faye ◽  
Ingolf F. Nes
Keyword(s):  
Selection Marker ◽  
Multiple Cloning Site ◽  
Propionibacterium Freudenreichii ◽  
Expression Plasmid ◽  
Physiochemical Properties ◽  
Food Grade ◽  
Cloning System ◽  
Immunity Gene ◽  
Membrane Bound ◽  
High Level

ABSTRACT This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

scholarly journals Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

2001 ◽  
Vol 67 (4) ◽  
pp. 1700-1709 ◽  
Author(s):  
Éric Émond ◽  
Richard Lavallée ◽  
Geneviève Drolet ◽  
Sylvain Moineau ◽  
Gisèle LaPointe
Keyword(s):  
Lactococcus Lactis ◽  
Resistance Mechanism ◽  
Laboratory Strain ◽  
Plasmid Profile ◽  
Selection Marker ◽  
Erythromycin Resistance ◽  
Food Grade ◽  
Cloning System ◽  
Two Component ◽  
Industrial Strains

ABSTRACT pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putativecis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-actingori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is arepB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis iftrans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactisand two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.

scholarly journals A new assay for nitric oxide reductase reveals two conserved glutamate residues form the entrance to a proton-conducting channel in the bacterial enzyme

2006 ◽  
Vol 401 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Faye H. Thorndycroft ◽  
Gareth Butland ◽  
David J. Richardson ◽  
Nicholas J. Watmough
Keyword(s):  
Nitric Oxide ◽  
Paracoccus Denitrificans ◽  
Nitric Oxide Reductase ◽  
Transmembrane Helices ◽  
Level Of Activity ◽  
Bacterial Enzyme ◽  
Cytoplasmic Membranes ◽  
Membrane Bound ◽  
Proton Movement ◽  
High Level

A specific amperometric assay was developed for the membrane-bound NOR [NO (nitric oxide) reductase] from the model denitrifying bacterium Paracoccus denitrificans using its natural electron donor, pseudoazurin, as a co-substrate. The method allows the rapid and specific assay of NO reduction catalysed by recombinant NOR expressed in the cytoplasmic membranes of Escherichia coli. The effect on enzyme activity of substituting alanine, aspartate or glutamine for two highly conserved glutamate residues, which lie in a periplasmic facing loop between transmembrane helices III and IV in the catalytic subunit of NOR, was determined using this method. Three of the substitutions (E122A, E125A and E125D) lead to an almost complete loss of NOR activity. Some activity is retained when either Glu122 or Glu125 is substituted with a glutamine residue, but only replacement of Glu122 with an aspartate residue retains a high level of activity. These results are interpreted in terms of these residues forming the mouth of a channel that conducts substrate protons to the active site of NOR during turnover. This channel is also likely to be that responsible in the coupling of proton movement to electron transfer during the oxidation of fully reduced NOR with oxygen [U. Flock, N. J. Watmough and P. Ädelroth (2005) Biochemistry 44, 10711–10719].

scholarly journals Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

2012 ◽  
Vol 78 (9) ◽  
pp. 3087-3097 ◽  
Author(s):  
Orla Condell ◽  
Carol Iversen ◽  
Shane Cooney ◽  
Karen A. Power ◽  
Ciara Walsh ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Food Industry ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Antimicrobial Compounds ◽  
Cross Tolerance ◽  
Content Type ◽  
Food Grade ◽  
High Level

ABSTRACTBiocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR)Salmonella entericastrains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds ofin vitroselection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure ofSalmonellastrains to an active biocidal compound, a high-level of tolerance was selected for a number ofSalmonellaserotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonicSalmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.

scholarly journals Glucose Signaling Pathway and Growth Conditions Regulate Gene Expression in Retrotransposon Ty2

2009 ◽  
Vol 64 (7-8) ◽  
pp. 526-532 ◽  
Author(s):  
Sezai Türkel ◽  
Özgür Bayram ◽  
Elif Arık
Keyword(s):  
Gene Expression ◽  
Growth Conditions ◽  
Glucose Sensors ◽  
Yeast Cells ◽  
Growth Stages ◽  
Regulate Gene Expression ◽  
Glucose Signaling ◽  
Yeast Cultures ◽  
Membrane Bound ◽  
High Level

Gene expression in the yeast retrotransposon Ty2 is regulated at transcriptional and translational levels. In this study, we have shown that the transcription of Ty2 is partially dependent on the membrane-bound glucose sensors Gpr1p and Mth1p in Saccharomyces cerevisiae. Transcription of Ty2 decreased approx. 3-fold in the gpr1, mth1 yeast mutant. Moreover, our results revealed that the transcription of Ty2 fluctuates during the growth stages of S. cerevisae. Both transcription and the frameshift rate of Ty2 rapidly dropped when the stationary stage yeast cells were inoculated into fresh medium. There was an instant activation of Ty2 transcription and a high level expression during the entire logarithmic stage of yeast growth. However, the transcription of Ty2 decreased 2-fold when the yeast cultures entered the stationary stage. The frameshift rate in Ty2 also varied depending on the growth conditions. The highest frameshift level was observed during the mid-logarithmic stage. It decreased up to 2-fold during the stationary stage. Furthermore, we have found that the frameshift rate of Ty2 diminished at least 5-fold in slowly growing yeasts. These results indicate that the transcription and the frameshift efficiency are coordinately regulated in the retrotransposon Ty2 depending on the growth conditions of S. cerevisiae.

scholarly journals Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen

2016 ◽  
Vol 84 (4) ◽  
pp. 1083-1091 ◽  
Author(s):  
Ryan McCormack ◽  
Wael Bahnan ◽  
Niraj Shrestha ◽  
Justin Boucher ◽  
Marcella Barreto ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Virulence Gene ◽  
Systemic Infection ◽  
Protective Role ◽  
Host Cells ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Membrane Bound ◽  
High Level ◽  
Macpf Domain

The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1,Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogenListeria monocytogenes. Within a few hours of systemic infection, the massive proliferation ofL. monocytogenesinPerforin-2−/−mice leads to a rapid appearance of acute disease symptoms. We go on to show in culturedPerforin-2−/−cells that the vacuole-to-cytosol transitioning ofL. monocytogenesis greatly accelerated. Unexpectedly, we found that inPerforin-2−/−macrophages,Listeria-containing vacuoles quickly (≤15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation ofL. monocytogenesto its replicative niche in the cytosol. This hypothesis was supported by our finding that aL. monocytogenesstrain expressing virulence factors at a constitutively high level replicated equally well inPerforin-2+/+andPerforin-2−/−macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification ofListeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.

scholarly journals Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wiggert J. Altenburg ◽  
N. Amy Yewdall ◽  
Daan F. M. Vervoort ◽  
Marleen H. M. E. van Stevendaal ◽  
Alexander F. Mason ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Nitrilotriacetic Acid ◽  
Binding Motif ◽  
Biologically Relevant ◽  
Enzyme Cascade ◽  
Synthetic Cell ◽  
Synthetic Cells ◽  
Membrane Bound ◽  
High Concentrations ◽  
High Level

AbstractThe cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

Stable expression plasmid for high-level production of GroE molecular chaperones in large-scale cultures

1993 ◽  
Vol 15 (9) ◽  
pp. 730-735 ◽  
Author(s):  
Cathy E. Kalbach ◽  
Anthony A. Gatenby
Keyword(s):  
Molecular Chaperones ◽  
Large Scale ◽  
Stable Expression ◽  
Expression Plasmid ◽  
High Level ◽  
Level Production

scholarly journals Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

2011 ◽  
Vol 78 (2) ◽  
pp. 568-574 ◽  
Author(s):  
Tao Zheng ◽  
Qihong Huang ◽  
Changyi Zhang ◽  
Jinfeng Ni ◽  
Qunxin She ◽  
...  
Keyword(s):  
Selectable Marker ◽  
Gene Knockout ◽  
Plasmid Vector ◽  
Selection Marker ◽  
Content Type ◽  
Sulfolobus Islandicus ◽  
Gene Coding ◽  
High Level ◽  
Coa Reductase

ABSTRACTWe report here a novel selectable marker for the hyperthermophilic crenarchaeonSulfolobus islandicus. The marker cassette is composed of thesac7dpromoter and thehmggene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Psac7d-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting thepyrEFgene of the expression vector pSeSD for Psac7d-hmgwith which theSulfolobusexpression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization ofSulfolobustransformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His6-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEFandhmg) was subsequently exploited for genetic analysis. AherAmerodiploid strain ofS. islandicuswas constructed usingpyrEFmarker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from theherAmerodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability ofS. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

scholarly journals Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria

2002 ◽  
Vol 68 (11) ◽  
pp. 5663-5670 ◽  
Author(s):  
Peter A. Bron ◽  
Marcos G. Benchimol ◽  
Jolanda Lambert ◽  
Emmanuelle Palumbo ◽  
Marie Deghorain ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Competitive Inhibitor ◽  
Selection Marker ◽  
Wild Type ◽  
Gene Encoding ◽  
Food Grade ◽  
Selection For ◽  
Isogenic Mutants ◽  
Type Strains

ABSTRACT Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of d-alanine and l-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Δalr) showed auxotrophy for d-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented d-alanine auxotrophy in the L. plantarum Δalr and L. lactis Δalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to d-cycloserine, a competitive inhibitor of Alr (600 and 200 μg/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that d-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Δalr. The resulting strain could grow in the absence of d-alanine only when expression of the alr gene was induced with nisin.

scholarly journals Development of a Novel Chloramphenicol Resistance Expression Plasmid Used for Genetic Complementation of a fliG Deletion Mutant in Treponema denticola

2004 ◽  
Vol 72 (9) ◽  
pp. 5493-5497 ◽  
Author(s):  
Linda L. Slivienski-Gebhardt ◽  
Jacques Izard ◽  
William A. Samsonoff ◽  
Ronald J. Limberger
Keyword(s):  
Treponema Pallidum ◽  
Treponema Denticola ◽  
High Level Expression ◽  
Expression Plasmid ◽  
Chloramphenicol Resistance ◽  
Expression Of Genes ◽  
Colony Spreading ◽  
High Level ◽  
Resistance Expression

ABSTRACT A new expression plasmid containing the fla operon promoter and a staphylococcal chloramphenicol resistance gene, was constructed to help assess the role of fliG in Treponema denticola motility. Deletion of fliG resulted in a nonmotile mutant with a markedly decreased number of flagellar filaments. Wild-type fliG genes from T. denticola and from Treponema pallidum were cloned into this expression plasmid. In both cases, the gene restored the ability of the mutant to gyrate its cell ends and enabled colony spreading in agarose. This shuttle plasmid enables high-level expression of genes in T. denticola and possesses an efficient selectable marker that provides a new tool for treponemal genetics.

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