The impact of acrylonitrile and bioaugmentation on the biodegradation activity and bacterial community structure of a topsoil.

The analysis of the bacterial community within the soil using DGGE showed acrylonitrile (ACN) could lead to the selection of significantly similar communities. Moreover, Rhodococcus sp. AJ270 was successfully established in the soil community. High GC G+-bacteria also responded positively to ACN addition. Bioaugmentation or carbon addition had no impact on the rate or degree of ACN degradation. ACN could be readily degraded by the soil bacteria, however, the community structure was significantly affected by its addition as well as by the addition of carbon or Rhodococcus sp. AJ270. The bioaugmentation of the soil with this strain was successful, in that the organism became established within the community. ACN addition to a soil produces statistically significant changes in the bacterial community.

Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells.

Although light microscopy fell out of favor as a research tool in prokaryotic biology in the 1980s, advances in the reagents available for cell labeling (staining) and in the user-friendliness of microscopes were underpinning a revolution in eukaryotic cell biology. The development of epifluorescence hardware, particularly confocal microscopy and low-light imaging systems, and computational deblurring and video enhancement methodologies, substantially extended the range of potential applications. These developments now enable us to detect weaker signals at higher levels of resolution than was previously possible. Finally the personal computer and related software developments have brought image analysis within affordable range for many laboratories and facilitate quantitation of cellular properties on an objective basis. We have sought to apply these advances across a range of prokaryotic applications and here we describe the methods we have applied to live Mycobacterium tuberculosis cells. Although we have principally been concerned with two applications, the determination of viability at the cellular level (see Note 1) and the nature and distribution of lipid domains, more general aspects of light microscopic cytological analyses are discussed below.

Lipid domains of mycobacteria studied with fluorescent molecular probes.

The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non-tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non-covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl-arabinogalactan-peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high-resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.