A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.

New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria.

Saccharomyces cerevisiae cell cycle.

https://www.cell.com/cell/pdf/0092-8674(92)90146-4.pdf

Ssn6-Tup1 is a general repressor of transcription in yeast.

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

/pdf/the-proteolytic-systems-of-lactic-acid-bacteria-1j6y0bndup.pdf

The proteolytic systems of lactic acid bacteria

Replacement of Escherichia coli's RecBCD function with phage lambda's Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate. The rate is at least 70-fold higher than that exhibited by a recBC sbcBC or recD strain. The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment. The deltarecBCD::Plac-red kan replacement allele can be P1 transduced to other E. coli strains, making the hyper-Rec phenotype easily transferable.

http://andrew-michaelson.com/Genetics/AdditionalPaperstoRead/MutantIsolationandAnalysis/9a.pdf

Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli.

A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

/pdf/a-general-system-for-generating-unlabelled-gene-replacements-53gzlc914u.pdf

A general system for generating unlabelled gene replacements in bacterial chromosomes

A character originating from Saccharomyces cerevisiae 1403-7A is described which interferes with maltose growth in the respiratory-deficient state. This character is inherited in an apparently non-Mendelian way, but at present no statement can be made concerning the localization of this character on a plasmid or the involvement of multiple genes. As a revertant of this character, a flaky mutant was isolated, showing a heavy flocculation during growth on liquid medium and resistance to catabolite repression for maltase, alpha-methyl-glucosidase, invertase, and succinate dehydrogenase. In wild-type cells, repression (caused by growth on 2% glucose) and derepression (caused by growth on 2% galactose) can be correlated with a lower and a higher level of cyclic 3',5'-adenosine monophosphate (cAMP), respectively. In cells of flaky mutant, growth on these carbon sources results in the same levels of cAMP as observed for the wild type. Consequently, in this mutant derepression in the presence of 2% glucose is not reflected in a higher level of cAMP.

Isolation of a catabolite repression mutant of yeast as a revertant of a strain that is maltose negative in the respiratory-deficient state.

We studied the glucose-6-phosphatase (G6Pase) gene of 30 unrelated glycogen storage disease type Ia (GSD Ia) patients using single strand conformational polymorphism (SSCP) prior to automated sequencing of exons revealing an aberrant SSCP pattern. In all patients we could identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure. A total of 14 different mutations were identified. R83C (16/60), 158delC (12/60), Q347X (7/60), R170X (6/60) and ΔF327 (4/60) were found most frequently. Nine other mutations accounted for the other 15 mutant alleles. Two DNA-based prenatal diagnoses were performed successfully. At present, 56 mutations in the G6Pase gene have been reported in 300 unrelated GSD Ia patients and an overview of these mutations is presented. Evidence for a clear genotype-phenotype correlation could be established neither from our data nor from those in the literature. With increased knowledge about the genetic basis of GSD Ia and GSD Ib and the high detection rate of mutations, it is our opinion that the diagnoses GSD Ia and GSD Ib can usually be based on clinical and biochemical abnormalities combined with mutation analysis instead of enzyme assays in liver tissue obtained by biopsy. A newly developed flowchart for the diagnosis of GSD I is presented. 
Conclusion Increased knowledge of the genetic basis of glycogen storage disease type I provides a DNA-based diagnosis, prenatal DNA-based diagnosis in chorionic villus samples and carrier detection.

Glycogen storage disease type Ia : recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flowchart

Summary0405 021)In addition to the mutant C2, described previously, 9 other constitutive MLA6-mutants have been isolated. These, like C2, are sensitive to catabolite repression and not super-inducible by maltose. One of these mutants (C19) is dominant relative to the wild type strain and to mal6-mutants. The others, like C2, are recessive to the wild type, and their hybrids with mal6-mutants show a partial or complete loss of the constitutivity and a partial or complete restoration of the inducibility, depending on the particular mutant combination used, which indicates that these recessive constitutive MAL6-mutations are allelic to the mal6-mutations. The mutation MAL6c19is possiblynot allelic to the mal6-mutations.2)A maltose negative mutation (malx) of a type different from the mal6-mutations was isolated. This mutation, unlinked to MAL6, is not expressed in combination with the wild type MAL6-gene, but only in the presence of constitutive MAL6c-alleles by changing the constitutive phenotype (not the genotype) into a mal6-phenotype. Similarly, another mutation (maly) was found that is linked neither to MAL6 nor to malxand that changes the phenotype of the constitutive allele MAL6c14into a mal6-phenotype. In contrast with malx, mutation malyis specific for the allele MAL6c14.3)The properties of the new constitutive mutants and the nature of the suppressors malxand malyand their importance for mapping constitutive MAL6c-alleles are discussed.

Regulation of maltose fermentation in Saccharomyces carlsbergensis. 3. Constitutive mutations at the MAL6-locus and suppressors changing a constitutive phenotype into a maltose negative phenotype.

A M ten Berge

Papers

A general system for generating unlabelled gene replacements in bacterial chromosomes

Isolation of a catabolite repression mutant of yeast as a revertant of a strain that is maltose negative in the respiratory-deficient state.

Glycogen storage disease type Ia : recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flowchart

Regulation of maltose fermentation in Saccharomyces carlsbergensis. 3. Constitutive mutations at the MAL6-locus and suppressors changing a constitutive phenotype into a maltose negative phenotype.

Regulation of maltose fermentation in Saccharomyces carlsbergensis. I. The function of the gene MAL6, as recognized by mal6-mutants.