pSEUDO, a Genetic Integration Standard for Lactococcus lactis

Pinto, Joao P. C.; Zeyniyev, Araz; Karsens, Harma; Trip, Hein; Lolkema, Juke S.; Kuipers, Oscar P.

doi:10.1128/aem.05196-11

Cited by 37 publications

(28 citation statements)

References 23 publications

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“…Three terminators downstream of the gfp gene terminate transcription and prevent read-through transcription from downstream genes. The regions of the pseudo 10 gene flanking the gfp gene facilitate integration at the pseudo 10 locus in the L. lactis chromosome (38). An erythromycin resistance cassette allows for selection in L. lactis.…”

Section: Resultsmentioning

confidence: 99%

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Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

Overkamp

Beilharz

Weme

et al. 2013

Appl Environ Microbiol

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bGreen fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two "superfolder" GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

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Section: Resultsmentioning

confidence: 99%

“…The gfpmut1 gene was amplified by PCR with primer pair gfp_F/gfp_R using pKB01_gfpmut1 as the template. The amplicon was inserted in L. lactis integration vector pSEUDO-GFP (38) as an XhoI/BamHI restriction fragment replacing the resident gfp-sf gene. This yielded the pSEUDOgfpmut1.…”

Section: Methodsmentioning

confidence: 99%

Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

Overkamp

Beilharz

Weme

et al. 2013

Appl Environ Microbiol

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“…The PCR products were self-ligated after digestion with restriction enzymes, resulting in the deletion constructs, which were transformed into E. coli DH5␣ for propagation. The plasmids were transformed into L. lactis JP9000, an MG1363 derivative containing the nisRK genes in the pseudo_10 locus (23). The procedure was continued as described above for the deletion of lysQ.…”

Section: Methodsmentioning

confidence: 99%

“…L. lactis NZ9000 (22) and JP9000 (23), strains derived from strain MG1363 carrying the nisRK genes in the pepN and pseudo_10 loci, respectively, were used for the nisin-inducible expression of amino acid transporter genes and as parental strains of transporter deletion mutants. L. lactis cells were grown at 30°C in M17 medium supplemented with 25 mM glucose (here referred to as GM17 medium) and containing 5 g/ml chloramphenicol, when appropriate, or in SA medium, a chemically defined medium (6) containing free amino acids, unless stated otherwise.…”

Section: Methodsmentioning

confidence: 99%

Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

2013

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Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.

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“…Lactococcus lactis JP9000, derived from strain MG1363 and carrying the nisRK genes in the pseudo_10 locus (20), was used as the parent for construction of the mutants containing the deletions ⌬serP1, ⌬serP2, and ⌬serP1P2. The ⌬serP1P2 double mutant was used as the host for the nisin-inducible expression of the SerP1 and SerP2 transporters in the P1⌬serP1P2 and P2⌬serP1P2 strains, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

Noens

Lolkema

2015

J Bacteriol

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The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports L-serine, L-threonine, and L-cysteine with high affinity. Affinity constants (K m ) are in the 20 to 40 M range. SerP2 is a DLalanine/DL-serine/glycine transporter. The preferred substrate appears to be DL-alanine for which the affinities were found to be 38 and 20 M for the D and L isomers, respectively. The common substrate L-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 M, respectively. Growth experiments demonstrate that SerP1 is the main L-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 substrates L-alanine and glycine. T he lactic acid bacterium (LAB) Lactococcus lactis is extensively used for the manufacture of buttermilk and cheese. L. lactis originally lived on plants, and dairy strains are currently derived by reductive evolution (1, 2). The change to nutrient-rich environments like milk is believed to be the reason that dairy strains of L. lactis lost biosynthetic pathways for various amino acids like branched-chain amino acids and histidine (3, 4). In milk, casein provides a rich source of all amino acids. It is degraded by an efficient proteolytic system, which consists of a proteinase, several peptidases, and a peptide transport system. The proteinase PrtP is attached extracellularly to the cell wall and hydrolyzes casein to peptides varying in length from 5 to more than 30 amino acid residues. The peptides are transported into the cell via the oligopeptide uptake system Opp and further degraded in the cell by intracellular peptidases (5, 6). The system generates free amino acids in the cytoplasm following uptake of peptides without the need for specific transporters to take up free amino acids from the environment. Nevertheless, media containing free amino acids as the sole source of amino acids do support growth of L. lactis (7), indicating that transport systems for single amino acids have been preserved. The physiological roles of many of these transporters during growth on different media are not clear.Transport studies in L. lactis and other LAB, mainly performed in the late 1980s, using whole cells or membrane vesicles demonstrated proton gradient-driven uptake activity for branched-chain amino acids and L-methionine (8, 9), L-alanine and glycine (10), L-lysine (11), L-serine, L-threonine, L-histidine, and L-cysteine (12), and L-tyrosine and L-phenylalanine (13). L-Glutamate, Lglutamine (14), and possibly L-proline (15) transport was shown to be driven by ATP hydrolysis. More recent work has identified many of the genes encoding the transporters. bcaP and gnlPQ were identified in L. lactis as encoding a secondary branche...

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pSEUDO, a Genetic Integration Standard for Lactococcus lactis

Cited by 37 publications

References 23 publications

Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

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

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