微生物学技术

In this chapter, we describe in detail the steps involved in isolation and characterization of lipoglycans from Mycobacterium tuberculosis and Mycobacterium smegmatis. In addition, procedures involved in structural analysis such as immunoblotting with mAb CS-35 or CS-40, gas chr ...

This chapter describes two protocols for isolating total RNA from mycobacteria: one for extraction from in vitro cultures and one for extraction from in vivo. In these protocols, RNA is liberated from mycobacteria by disruption with small glass beads in the presence of Trizol to stabilize the R ...

Environmental amoebae have been shown to be a host to pathogenic mycobacteria. Mycobacterium avium, Mycobacterium marinum, and Mycobacterium peregrinum can all grow inside Acanthamoeba and other environmental amoebae. Once ingested by Acanthamoeba, M. avium upregulates a num ...

MycoDB (http://myco.bham.ac.uk) is an online resource designed to facilitate genomic analyses of Mycobacterium spp. and related genera. Regions of interest can be found by searching the annotation, BLAST searching against the sequence data, or specifying genomic coordinates. Tools ...

Phage transduction is an attractive method of genetic manipulation in mycobacteria. ΦMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an ...

The ability to select genes to knock out of mycobacterial genomes has greatly improved our understanding of mycobacteria. This chapter describes a method for doing this. The gene (including a 1-kb flanking region) is cloned into a pNIL series vector and disrupted by deletion or insertion of a cass ...

A myriad of methods has been reported for the isolation of genomic DNA from Mycobacterium spp.; some methods use mechanical disruption of the bacterial cells, whereas others use some form of chemical or enzymatic lysis. Regardless of the approach, the end points remain efficient breaking of the ...

Site-specific recombinases such as the Saccharomyces cerevisiae Flp and the P1 phage Cre proteins have been increasingly used for the construction of unmarked deletions in bacteria. Both systems consist of an antibiotic resistance gene flanked by recognition sites in direct orient ...

The ability of bacteria to survive in a variety of different niches is due, in part, to their ability to respond and adapt to the environment. Extracellular signals are recognized by bacilli, and their responses are generally conducted at the transcript level. RNA polymerases recognize speci ...

A wide variety of inducible expression systems have been designed for Gram-negative bacteria, but adapting these systems to phylogenetically distinct species, such as mycobacteria, has proved notoriously difficult. Mycobacteria belong to a class of high G+C Gram-positive bacte ...

Elucidating the function of mycobacterial proteins, determining their contribution to virulence, and developing new vaccine candidates has been facilitated by systems permitting the heterologous expression of genes in mycobacteria. Mycobacterium bovis bacille Calme ...

The construction of allelic exchange mutants is one of the major strategies to decipher the function of a defined gene. In this chapter, protocols are described to perform allelic exchange in the mycobacterial species Mycobacterium smegmatis and Mycobacterium tuberculosis using th ...

The importance of plasmids for molecular research cannot be underestimated. These double-stranded DNA units that replicate independently of the chromosomal DNA are as valuable to bacterial geneticists as a carpenter’s hammer. Fortunately, today the mycobacterial research co ...

With the increased need for novel antimicrobials to improve the existing treatment for tuberculosis, to combat multidrug-resistant tuberculosis, and to address the presence of latent bacilli in a large population throughout the world, which can reactivate and cause active disease, ...

An agar dilution method for measuring minimum inhibitory concentrations (MICs) of Mycobacterium tuberculosis, based on the method of proportion, is described. Mycobacterium strains are grown on Middlebrook 7H10 (or 7H11) agar medium with twofold serially diluted drug concentr ...

Batch cultures have predominately been used for the study of physiology and gene expression in mycobacteria. This chapter describes the assembly of chemostats and the methodology that is being used for growing Mycobacterium tuberculosis in continuous culture, which provides the gr ...

The oral cavity harbors several hundred different bacterial species that colonize both hard (teeth) and soft tissues, forming complex populations known as microbial biofilms. It is widely accepted that the phenotypic characteristics of bacteria grown in biofilms are substantia ...

In their natural environments, microorganisms usually live in organized communities. Profiling analysis of microbial communities has recently assumed special relevance as it allows a thorough understanding of the diversity of the microbiota, its behavior over time, and the esta ...

Application of nucleic acid technology to the analysis of the bacterial diversity in the oral cavity in conditions of health and disease has not only confirmed the findings from early culture studies but also significantly expanded the list of oral inhabitants and candidate pathogens ass ...

Azole antifungal drugs are used widely to treat people with oral fungal infections. Unfortunately, fungi can develop resistance to these drugs. This resistance can be due to the overexpression or mutation of cytochrome P450 14α-lanosterol demethylase, also known as ERG11 or CYP51, and/or ...