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PubMed2001

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-motile mutants deficient in endoflagella.

Leptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin-resistant colonies resulting from single and double cross-over events at the flaB locus. The double recombinant mutants are non-motile, as visualized in both liquid and semi-solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycin-resistant strain. Like the flaB mutant studied above, the Strr double cross-over mutant was non-motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.

Alleles↗
PubMed1999

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Biological Evolution↗
PubMed1995

Effects of dietary fat on high-density-lipoprotein subclasses are influenced by both apolipoprotein E isoforms and low-density-lipoprotein subclass patterns.

We examined the effects of replacing dietary fat with carbohydrates on high-density-lipoprotein (HDL) subclasses as measured by nondenaturing polyacrylamide-gradient-gel electrophoresis. One hundred five men received a 6-wk low-fat diet (24% of total energy) and a 6-wk high-fat diet (46% of energy) in a crossover design. Absorbency of protein stain was measured within five HDL subclasses: HDL3c (7.2-7.8 nm), HDL3b (7.8-8.2 nm), HDL3a (8.2-8.8 nm), HDL2a (8.8-9.7 nm), and HDL2b (9.7-12 nm). The low-density-lipoprotein-(LDL) subclass pattern was determined by gradient-gel electrophoresis, with pattern B men defined as having an LDL-predominant peak diameter < or = 25.5 nm and an LDL distribution skewed toward larger size particles. On the high-fat diet, 18 men exhibited LDL-subclass pattern B and 87 men exhibited the alternative LDL pattern A. Twelve men had the apolipoprotein (apo) epsilon 2 allele. Replacing dietary fat with carbohydrates 1) significantly decreased HDL3a, HDL2a, and HDL2b; 2) reduced HDL2b significantly more in pattern A than in pattern B men; and 3) increased plasma HDL3b concentrations significantly more in those men with the epsilon 2 allele. Our results suggest that unfavorable HDL changes were significantly more likely to occur in men who had LDL-subclass pattern A or the apo epsilon allele than in men who had pattern B or lacked the epsilon 2 allele.

Apolipoproteins E↗
PubMed2001

The opdB locus encodes the trypsin-like peptidase activity of Treponema denticola.

High levels of Treponema denticola in subgingival dental plaque are associated with severe periodontal disease. T. denticola, along with Porphyromonas gingivalis and Bacteroides forsythus, are the only cultivatable oral microorganisms that produce significant amounts of "trypsin-like" peptidase activity. The ability of subgingival plaque to hydrolyze N-alpha-benzoyl-DL-arginine-2-naphthylamide (BANA) is associated with high levels of one or more of these organisms. The purpose of this study was to identify the gene encoding trypsin-like activity in T. denticola and thus facilitate molecular-level studies of its potential role in disease. Using published peptide sequences of a T. denticola surface-associated oligopeptidase with BANA-hydrolyzing activity, we identified the gene, designated opdB, in an apparently noncoding region of the T. denticola genome unannotated contigs (11/2000; http://www.tigr.org). The opdB gene begins with a TTG start codon and encodes a 685-residue peptide with high homology to the oligopeptidase B family in prokaryotes and eukaryotes. An isogenic T. denticola opdB mutant was constructed by allelic replacement mutagenesis using an ermF/AM gene cassette. The mutant lacked BANA-hydrolyzing activity and had a slightly slower growth rate than the parent strain. This mutant will be used in future studies of interactions of T. denticola with host cells and tissue.

Alleles↗
PubMed1994

Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii.

To investigate the feasibility of genomic transgene expression and gene targeting in Toxoplasma gondii, parasites have been transfected with constructs differing in the length of contiguous genomic sequence spanning the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously reported that vectors derived from a DHFR-TS cDNA 'minigene' containing mutations in the DHFR coding sequence confer pyrimethamine resistance to transfected parasites (Donald and Roos, 1993). Stably resistant parasite clones arise at high frequency, generally by virtue of transgene integration into parasite chromosomes at locations scattered throughout the genome. In contrast, using a vector which contains 8 kb of contiguous genomic sequence (vs. < 2 kb for the cDNA-derived vectors), approximately half of the integration events occur by homologous recombination. Homologous recombination appears to occur at even higher frequency when a 16 kb genomic clone is used. Circular plasmids were more efficient than linearized molecules at producing homologous recombination in this system, integrating by reciprocal crossing-over to produce a duplication of the DHFR-TS locus. Double crossing-over (or gene conversion) was also observed at low frequency, resulting in complete allelic replacement in this haploid stage of the parasite. The ability to produce either homologous or non-homologous recombinants, by the selection of appropriate transformation constructs, has considerable genetic potential.

Alleles↗
PubMed2004

A common cross-species function for the double epidermal growth factor-like modules of the highly divergent plasmodium surface proteins MSP-1 and MSP-8.

An understanding of structural and functional constraints on the C-terminal double epidermal growth factor (EGF)-like modules of merozoite surface protein (MSP)-1 and related proteins is of importance to the development of these molecules as malaria vaccines and drug targets. Using allelic replacement, we show that Plasmodium falciparum parasites can invade erythrocytes and grow efficiently in the absence of an MSP-1 protein with authentic MSP-1 EGF domains. In this mutant parasite line, the MSP-1 EGFs were replaced by the corresponding double EGF module from P. berghei MSP-8, the sequence of which shares only low identity with its MSP-1 counterpart. Hence, the C-terminal EGF domains of at least some Plasmodium surface proteins appear to perform the same function in asexual blood-stage development. Mapping the surface location of the few residues that are common to these functionally complementary EGF modules revealed the presence of a highly conserved pocket of potential functional significance. In contrast to MSP-8, an even more divergent double EGF module, that from the sexual stage protein PbS25, was not capable of complementing MSP-1 EGF function. More surprisingly, two chimeric double EGF modules comprising hybrids of the EGF domains from P. falciparum and P. chabaudi MSP-1 were also not capable of replacing the P. falciparum MSP-1 EGF module. Together, these data suggest that although the MSP-1 EGFs can accommodate extensive sequence diversity, there appear to be constraints that may restrict the simple accumulation of point mutations in the face of immune pressure in the field.

Alleles↗
PubMed1991

Nucleotide sequence of the Escherichia coli regulatory gene mprA and construction and characterization of mprA-deficient mutants.

In high copy number, the Escherichia coli mprA gene reduces the synthesis of peptide microcins B17 and C7 (MccB17 and MccC7) and blocks the osmoinduction of the proU operon at the transcriptional level. mprA has been sequenced and shown to encode a polypeptide of 176 amino acids (Mr, 20,563). Insertion and deletion mutant mprA alleles were constructed and then transferred to the chromosome by allelic replacement. In these mutants, expression of two mcb-lacZ fusions was fivefold derepressed, indicating a negative regulatory role of mprA on the mcb operon (MccB17). In contrast, no effect of the MprA- mutations on the expression of mcc operon (MccC7) or on the osmoinduction of proU operon was observed.

Alleles↗
PubMed2004

Transfection of the human malaria parasite Plasmodium falciparum.

Methods to transiently and stably transfect blood stages of the human malaria parasite Plasmodium falciparum have been developed and adapted for gene-knockout, allelic replacement, and transgene expression in this organism. These methods are detailed in this chapter, as are approaches used to monitor transfectants during the selection process. The different plasmid vectors that are currently used for gene targeting and transgene expression (including green fluorescent protein expression) are also described.

Alleles↗
PubMed1998

Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties.

A gene encoding a novel cell wall-associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N-acetyl-muramyl-L-alanine amidase and for an endo-beta-N-acetyl-D-glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N-terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram-positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.

Adhesins, Bacterial↗
PubMed2001

Lack of fbe, the gene for a fibrinogen-binding protein from Staphylococcus epidermidis, reduces its adherence to fibrinogen coated surfaces.

The significance of Fbe, a fibrinogen-binding protein in Staphylococcus epidermidis, was investigated. A fbe mutant was constructed by allelic replacement, where a Gentamicin resistance gene replaced a portion of the A region of fbe. Adherence assay to immobilized fibrinogen on polyethylene surfaces and peripheral venous catheters from patients showed that the fibrinogen binding ability of the mutant was reduced compared to its parental strain. This shows that Fbe is a major factor involved in adherence of S. epidermidis to fibrinogen. No difference was found between the wild-type and mutant in their affinity to immobilized fibronectin.

Alleles↗
PubMed2000

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

Acetyl-CoA C-Acetyltransferase↗
PubMed1986

Chromosomal map location of the alpha-hemolysin structural gene in Staphylococcus aureus NCTC 8325.

The alpha-hemolysin structural gene, hly+, previously cloned, insertionally inactivated, and introduced into the chromosome by allele replacement (M.O. O'Reilly, J.C.S. De Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathog. 1:125-138, 1986), was shown by protoplast fusion and transformation to be in the gene order purC-hly-uraB-omega[chr::Tn916]1101-thrB on the chromosome of Staphylococcus aureus NCTC 8325. This location is clearly distinct from that of the agr determinant, a regulatory gene affecting several extracellular proteins, including alpha-hemolysin, located between tmn and ilv.

Alleles↗
PubMed1996

Progression of coronary atherosclerosis is associated with a common genetic variant of the human stromelysin-1 promoter which results in reduced gene expression.

There is a common polymorphism in the promoter sequence of the human stromelysin-1 gene, with one allele having a run of six adenosines (6A) and the other five adenosines (5A). We have previously reported, in a 3-year follow-up study of patients with coronary atherosclerosis, that those patients who are homozygous for the 6A allele show a more rapid progression of the disease. In this study, we have investigated whether the 5A/6A promoter polymorphism plays a role in the regulation of stromelysin-1 gene expression. In transient transfection experiments, a stromelysin-1 promoter construct with 6A at the polymorphic site was found to express less of the chloramphenicol acetyltransferase reporter gene than a construct containing 5A. Electrophoretic mobility shift assay and DNase I footprinting revealed the interaction of one or more nuclear protein(s) with the DNA sequence at the 5A/6A polymorphic site. The binding of one of the nucleoprotein factors was more readily detectable with an oligonucleotide probe corresponding to the 6A allele as compared with a probe corresponding to the 5A allele. Replacing the core binding sequence with a random DNA sequence abolished the interaction between the nuclear protein(s) and the probe and also increased reporter gene expression in transiently transfected cells. Thus, the common 5A/6A polymorphism of the human stromelysin-1 promoter appears to play an important role in regulating stromelysin-1 gene expression and may be involved in the progression of coronary heart disease.

Alleles↗
PubMed1990

Generation of capsule-deficient Neisseria meningitidis strains by homologous recombination.

Capsule-deficient mutants of Neisseria meningitidis serogroup B strain B1940 were constructed by allelic replacement using the plasmids pMF120 and pMF121, which contain the flanking regions of the gene locus for the biosynthesis pathway of the group B meningococcal capsular polysaccharide. Southern blot analysis of chromosomal DNA of the capsule-deficient meningococcal strains confirmed the generation of large deletions in the chromosomal cps gene complex. The same strategy proved useful in constructing meningococcal strains with capsular types A, C, W135, Y and Z.

Alleles↗
PubMed1999

Molecular and genetic analyses of Actinomyces spp.

Members of the genus Actinomyces are predominant primary colonizers of the oral cavity and play an important role in initiating plaque development. These bacteria have evolved unique mechanisms that favor colonization and persistence in this micro-environment. The expression of cell-surface fimbriae is correlated with the ability of these bacteria to adhere to specific receptors on the tooth and mucosal surfaces, and to interact with other plaque bacteria. The elaboration of sialidase is thought to enhance fimbriae-mediated adherence by unmasking the fimbrial receptors on mammalian cells. The presence of certain cell-associated or extracellular enzymes, including those involved in sucrose or urea metabolism, may provide the means for these bacteria to thrive under conditions when other growth nutrients are not available. Moreover, these enzyme activities may influence the distribution of other plaque bacteria and promote selection for Actinomyces spp. in certain ecological niches. The recent development of a genetic transfer system for Actinomyces spp. has allowed for studies the results of which demonstrate the existence of multiple genes involved in fimbriae synthesis and function, and facilitated the construction of allelic replacement mutants at each gene locus. Analyses of these mutants have revealed a direct correlation between the synthesis of assembled fimbriae and the observed adherence properties. Further genetic analysis of the various enzyme activities detected from strains of Actinomyces should allow for an assessment of the role of these components in microbial ecology, and their contribution to the overall success of Actinomyces spp. as a primary colonizer and a key player in oral health and disease.

Actinomyces↗
PubMed1990

The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants.

The sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325-4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N-terminal 150-270 residues, which are c. 50% identical, (ii) a central region with high (greater than 90%) residue identities, and (iii) a C-terminal region composed of repeated 27-amino-acid residue sequences. The variable N-terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N-terminal half of the protein. A site-specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele-replacement in strains 8325-4 and M60. The Coa- mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or nontoxigenic strains was obtained. This contradicts findings of several groups using Coa- mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence-related genes.

Alleles↗
PubMed1996

An ideR mutant of Mycobacterium smegmatis has derepressed siderophore production and an altered oxidative-stress response.

The mycobacterial ideR protein is a homologue of the diphtheria-toxin repressor DtxR. We have previously demonstrated that Mycobacterium tuberculosis ideR, like DtxR, represses transcription of Corynebacterium diphtheriae iron-regulated promoters in vivo and binds to C. diphtheriae operators in a metal-dependent manner in vitro. We show here that ideR mutants of M. smegmatis, constructed by allelic replacement, were defective in their ability to repress siderophore biosynthesis in the presence of iron. They were also more sensitive to hydrogen peroxide and had decreased levels of catalase/peroxidase (KatG) and manganese superoxide dismutase (Mn-SOD). This indicates that ideR is a negative regulator of siderophore production and is required for the response to superoxide- and hydrogen peroxide stress. We propose that ideR is the mycobacterial counterpart of the Escherichia coli Fur protein, i.e. It is a pleiotropic regulator that couples iron metabolism to the oxidative-stress response.

Alleles↗
PubMed1993

Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients.

Chronic respiratory infections with mucoid Pseudomonas aeruginosa are the leading cause of high mortality and morbidity in cystic fibrosis (CF). The initially colonizing strains are nonmucoid, but in the CF lung they invariably convert into the mucoid, exopolysaccharide alginate-overproducing form causing further deterioration and poor prognosis. Here we report the molecular basis of conversion to mucoidy. The algU gene is required for expression of the key alginate biosynthetic gene algD and encodes a protein homologous to sigma H, an alternative sigma factor regulating sporulation and other post-exponential-phase processes in Bacillus. The algU gene and the negative regulators mucA and mucB constitute the gene cluster controlling conversion to mucoidy. We demonstrate a critical role of mucA in this process based on (i) the presence of frameshift mutations disrupting the mucA coding region in mucoid cells that were absent in nonmucoid parental strains, (ii) genetic complementation of mucA mutations with the mucA+ gene, (iii) allelic replacements with specific mutant mucA genes causing conversion to mucoidy in previously nonmucoid cells, and (iv) detection of identical and additional mucA mutations in clinical mucoid strains isolated from the lungs of CF patients. These results suggest that the switch from the nonmucoid to mucoid state can be caused by inactivation of mucA, resulting in constitutive expression of alginate biosynthetic genes dependent on algU for transcription and that such mutants may be selected in vivo during chronic infections in CF.

Alginates↗