The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast- slow-growing mycobacteria. In this procedure gene of interest, disrupted by selectable marker, is cloned into conditionally replicating (temperature-sensitive) shuttle phasmid to generate specialized transducing mycobacteriophage. temperature-sensitive mutations the mycobacteriophage genome permit replication at permissive temperature 30 °C but prevent non-permissive 37 °C....

Mycobacteria produce beta-lactamases and are intrinsically resistant to beta-lactam antibiotics. In addition the beta-lactamases, cell envelope permeability variations in certain peptidoglycan biosynthetic enzymes believed contribute resistance these organisms. To allow study of additional mechanisms, mutants major BlaC BlaS, were generated pathogenic Mycobacterium tuberculosis strain H37Rv model organism smegmatis PM274. The M. PM638 (DeltablaC1) PM759 (DeltablaS1) showed an increase...

The peptidoglycan of most bacteria consists a repeating disaccharide unit β-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. However, the muramic moieties mycobacterial are N-glycolylated, not N-acetylated. This is rare modification seen only in mycobacteria five other closely related genera bacteria. N-glycolylation sialic acids unique carbohydrate that has been studied extensively eukaryotes. significance bacterial unknown. goal this project was to identify gene encoding...

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent was hindered by a lack methods for gene disruptions and allelic exchange. Several groups have described different disrupting genes marked with antibiotic resistance determinants in slow-growing organisms bovis bacillus Calmette-Guérin (BCG) M. tuberculosis. In this study, we first report using mycobacterial suicidal plasmid bearing counterselectable marker sacB exchange unmarked deletion mutations chromosomes...

Diaminopimelate (DAP) is a unique metabolite used for both the biosynthesis of lysine in bacteria and construction peptidoglycan many species bacteria, including mycobacteria. DAP synthesized by as part aspartate amino acid family, which includes methionine, threonine, isoleucine, lysine. Aspartokinase, first enzyme this pathway, encoded ask gene Previous attempts to disrupt Mycobacterium smegmatis were unsuccessful, even when cells supplied with all members suggesting that unlike other...

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas ability perform allelic exchanges has provided a useful method gene disruption in other organisms, clinically important species mycobacteria, such as M. and bovis, similar approaches thus far unsuccessful. In this communication, we report development shuttle mutagenesis strategy that involves use long linear recombination substrates reproducibly...

ABSTRACT The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for proper extracytoplasmic localization of involved in a variety cellular functions, including pathogenesis. Mycobacterium tuberculosis smegmatis genomes contain open reading frames with homology to components Tat export system (TatABC) as well potential Tat-exported possessing N-terminal signal sequences characteristic motif. Due importance exported...

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand internal pressure of bacteria. Bacteria are known heavily remodel their peptidoglycan stem peptides, yet little about physiological impact these chemical variations on cross-linking. Furthermore, there limited tools study structural variations, which can also have important implications cell wall integrity and host immunity. Cross-linking peptide chains within PG an essential process, its...

The polysialic acid capsule of Escherichia coli K1, a causative agent neonatal septicemia and meningitis, is an essential virulence determinant. 17-kb kps gene cluster, which divided into three functionally distinct regions, encodes proteins necessary for polymer synthesis expression at the cell surface. central region, 2, products required synthesis, activation, polymerization sialic acid, while flanking 1 3, are thought to be involved in assembly transport. In this study, we identified two...

ABSTRACT The processing of lipoproteins (Lpps) in Gram-negative bacteria is generally considered an essential pathway. Mature these are triacylated, with the final fatty acid addition performed by Lnt, apolipoprotein N-acyltransferase. mature then sorted Lol system, most Lpps inserted into outer membrane (OM). We demonstrate here that lnt gene not to pathogen Francisella tularensis subsp. strain Schu or live vaccine LVS. An LVS Δ mutant has a small-colony phenotype on sucrose medium and...

Escherichia coli has five genes encoding L,D-transpeptidases (Ldt) with varied functions. Three of these enzymes (YbiS, ErfK, YcfS) have been shown to cross-link Braun's lipoprotein the peptidoglycan (PG), while other two (YnhG, YcbB) form direct meso-diaminopimelate (DAP-DAP, or 3-3) cross-links within PG. In addition, Ldt can also incorporate non-canonical D-amino acids, such as D-methionine, into To further investigate role and, in particular, 3-3 linkages cell envelope physiology we...

Deep tissue abscesses are inflammatory, purulent lesions encased in a fibrin-rich pseudocapsule that include multiple bacterial and fungal species. We have initiated Phase 1 clinical trial exploring the safety feasibility of methylene blue photodynamic therapy (MB-PDT) at time abscess drainage. To optimize treatment parameters for future applications, our goal is to generate physically accurate three-dimensional (3D) models upon which bacteria can be grown. Here, we report results MB-PDT...

This paper is the first detailed description of development and use new genetic tools specifically for safe manipulation highly pathogenic Francisella tularensis subsp. tularensis. Most these are also demonstrated to work with other F. subspecies. Kanamycin hygromycin resistance determinants that function as markers in strain Schu sets episomal shuttle vectors either unstable or stably maintained absence selection were developed. In addition, hyg gene, expressed from groESL promoter, was...

ABSTRACT The in vivo growth phenotype and vaccine efficacy of a lysine auxotrophic mutant Mycobacterium tuberculosis strain H37Rv are described. An immunization experiment using mouse model with an aerosol challenge showed that two doses the M. were required to generate protection equivalent bovis BCG vaccine.

Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of biology and molecular pathogenesis F. has been limited by difficulties in manipulating such highly pathogenic organism lack genetic tools. However, recent advances have substantially improved ability researchers to genetically manipulate this organism. To expand toolbox we developed two systems stably integrate elements single-copy into genome. The first system...

l,d-Transpeptidases (Ldts) catalyse the formation of 3-3 cross-links in peptidoglycans (PGs); however, role these enzymes cell envelope physiology is not well understood. Mycobacterial PG contains a higher percentage (~30-80 %) than most other bacteria, suggesting that they are particularly important to mycobacterial wall biology. The genomes Mycobacterium tuberculosis and smegmatis encode multiple Ldt genes, but it clear if redundant. We compared sequences proteins from 18 found can be...

The Mycobacterium tuberculosis lysAgene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5′-phosphate (PLP)-dependent enzyme. catalyzes final step in lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) tol-lysine. lysA gene of M. H37Rv has been established as essential for bacterial survival immunocompromised mice, demonstrating thatde novo biosynthesis is vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and...

Our laboratory previously constructed mutants of Mycobacterium tuberculosis and smegmatis with deletions in the genes for their major beta-lactamases, BlaC BlaS, respectively, showed that have increased susceptibilities to most beta-lactam antibiotics, particularly penicillins. However, there is still a basal level resistance certain penicillins, some cephalosporin-based beta-lactams are essentially same as those wild types. We hypothesized characterizing additional (derived from...

ABSTRACT Class A penicillin-binding proteins (PBPs) are large, bifunctional that responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to only two class PBPs, PonA1 PonA2, encoded their genomes. We report here genomes smegmatis other soil mycobacteria contain an additional gene encoding a third protein, PonA3, which is paralog PonA2. Both PonA2 PonA3 p enicillin-binding protein nd s erine/ t hreonine...

The 17-kilobase kps gene cluster of Escherichia coli K1 contains all the information necessary for expression capsular polysaccharide.Region 3 encodes two genes, kpsM and kpsT, whose products belong to ATP-Binding Cassette (ABC)-transporter protein family.The KpsMT system is involved with export polysaccharide in E. coli.Earlier work indicated that interaction between KpsT ATP important transport.In this study, we report KpsT, a peripheral inner membrane protein, can be photolabeled by...

Abstract Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success the ability bacteria to infect immunocompetent individuals and evade eradication by adaptive immune response that includes production macrophage-activating cytokine, IFN-γ. Although IFN-γ essential for arrest progressive tuberculosis, it insufficient efficacious macrophage killing bacteria, which may be due M. inhibit selected responses In vitro studies have determined...

Abstract Objectives The objective of this study was to determine the effects methylene blue (MB) concentration, laser fluence rate, and on efficacy in vitro photodynamic therapy (PDT) for four bacteria commonly found human abscesses. Materials Methods PDT experiments were performed with most common abdominal abscesses: Escherichia coli ( E. ), Enterococcus faecalis Staphylococcus aureus S. Pseudomonas aeruginosa P. ). MB concentration varied from 50-300 µg/mL, rate 1-4 mW/cm 2 at a 7.2 J/cm...

The objective of this study was to determine the effects methylene blue (MB) concentration, laser fluence rate, and on efficacy in vitro photodynamic therapy (PDT) for four bacteria commonly found human abscesses. PDT experiments were performed with most common abdominal abscesses: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa. MB concentration varied from 50 300 μg/mL, rate 1 4 mW/cm2 at a 7.2 J/cm2. Higher rates fluences explored P. Primary outcomes...