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Leenhouts, Kees; Venema, Gerard

doi:10.1023/a:1009862119114

Cited by 24 publications

(7 citation statements)

References 28 publications

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“…Plasmid pCL49 was obtained by insertion of the 2-kb plsX (truncated) plus fabH PCR product of S. enterica chromosomal DNA (amplified with primers SalS-N and SalH-C, Table II) into pCR2.1 (Invitrogen). Plasmid pORI280 that contains an erythromycin resistance gene, the origin of lactococcal replication of plasmid pWV01, and the E. coli ␤-galactosidase gene expressed under lactococcal promoter P 32 was used as the vector for gene replacement (19). Plasmid pCL58 was constructed by insertion of the 1.2-kb fabH PCR product of L. lactis chromosome DNA (amplified with primers LacH-P and LacH-C, Table II) into pCR2.1.…”

Section: Methodsmentioning

confidence: 99%

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β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is Essential for Bacterial Fatty Acid Synthesis

Lai

Cronan

2003

Journal of Biological Chemistry

160

144

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␤-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids. However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated. We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403. The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids. The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism. However, the L. lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required. Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation (which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain. Indeed, incorporation of [1-14 C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers. We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system. Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.Fatty acid biosynthetic pathways are of two classes called types I and II (reviewed in Refs. 1 and 2). In the associated or type I fatty acid synthase system, each fatty acid synthetic reaction is catalyzed by a distinct domain of large multifunctional proteins. The dissociated or type II fatty acid synthesis system found in most bacteria and plant plastids consists of a series of discrete proteins, each of which catalyzes an individual reaction of the fatty acid biosynthetic pathway. In some cases, two or more enzymes are able to perform the same chemical reaction, but have differing substrate specificities and physiological functions. The ...

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Section: Methodsmentioning

confidence: 99%

“…L. lactis cultures were grown in GM17 medium (Difco) or on GM17 agar plates (19). Antibiotics were added at the following concentrations (in g/ml): kanamycin, 50; ampicillin, 100; spectinomycin, 100; tetracycline, 12; and erythromycin, 150.…”

Section: Methodsmentioning

confidence: 99%

β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is Essential for Bacterial Fatty Acid Synthesis

Lai

Cronan

2003

Journal of Biological Chemistry

160

144

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“…To construct the L. lactis lspA mutant strain MG1363⌬lsp, the plasmid pORI280-based chromosomal integration-excision system developed by Leenhouts et al (29,41) was used. For this purpose, two fragments of the lspA region were amplified by PCR.…”

Section: Methodsmentioning

confidence: 99%

Active Lipoprotein Precursors in the Gram-positive EubacteriumLactococcus lactis

Venema¹,

Tjalsma²,

Dijl³

et al. 2003

Journal of Biological Chemistry

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Lipid-modified proteins play important roles at the interface between eubacterial cells and their environment. The importance of lipoprotein processing by signal peptidase II (SPase II) is underscored by the fact that this enzyme is essential for viability of the Gramnegative eubacterium Escherichia coli. In contrast, SPase II is not essential for growth and viability of the Gram-positive eubacterium Bacillus subtilis. This could be due to alternative amino-terminal lipoprotein processing, which was shown previously to occur in SPase II mutants of B. subtilis. Alternatively, uncleaved lipoprotein precursors might be functional. To explore further the importance of lipoprotein processing in Gram-positive eubacteria, an SPase II mutant strain of Lactococcus lactis was constructed. Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are essential for growth in milk, the growth of SPase II mutant L. lactis cells in this medium was not affected. Furthermore, the activity of the strictly PrtMdependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II. Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase II. Taken together, these findings show for the first time that authentic lipoprotein precursors retain biological activity.

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“…Many tools are now available for genetic modification of L. lactis [38][39][40]. Plasmid vectors have been constructed for the cloning of genes.…”

Section: Genetic Engineering Of Lactococcus Lactismentioning

confidence: 99%

“…The most versatile of several different insertion vectors, one based on the replicon of pWV01 [40], will be described in some more detail here to enable deeper treatment of the creation of calculated chromosomal mutations.…”

Section: Genetic Engineering Of Lactococcus Lactismentioning

confidence: 99%

Mutations in Lactococcus lactis and their Detection

Kok¹,

Burg²

2006

Genetically Engineered Food

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Many methods are available for permanent alteration or mutation, at will, of the genetic make-up of Lactococcus lactis. Strains with different properties can be selected from natural or industrial environments or can be isolated after application of a variety of classical mutation strategies to existing strains. In the last two decades we have seen the rapid development of sophisticated genetic engineering techniques for application to L. lactis. Recombinant DNA technology has advanced to such perfection that it is now, in principle, possible to introduce any mutation, small or large, or to insert genes from any origin into the genome of L. lactis. These possibilities open up a wide array of new applications of L. lactis, in food or feed production or for entirely new (medical) applications. In this review we describe in detail the potential of altering the genetic make up of L. lactis, by classical techniques and by recombinant DNA technology. We will examine the possibilities of distinguishing the strains made by the latter techniques, so-called genetically modified organisms, from "natural" mutants and will discuss methods for detection of genetically modified strains of L. lactis. IntroductionGenetically modified organisms (GMOs) are organisms in which permanent DNA alterations have been introduced by recombinant DNA techniques. Over the last two decades, Lactococcus lactis has been made amenable to recombinant DNA technology. Tools to transform L. lactis, to introduce and express (foreign) DNA, to secrete (heterologous) proteins, and to mutate the chromosome by single and double crossover recombination strategies have all been developed. Constitutive or regulated promoters can be used to drive (foreign) gene expression. The culmination of genetic dissection of L. lactis was the determination in 2001 of the entire nucleotide sequence of the chromosome of L. lactis subsp. Lactis IL1403 [1a].

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Untitled

Cited by 24 publications

References 28 publications

β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is Essential for Bacterial Fatty Acid Synthesis

β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is Essential for Bacterial Fatty Acid Synthesis

Active Lipoprotein Precursors in the Gram-positive EubacteriumLactococcus lactis

Mutations in Lactococcus lactis and their Detection

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