Overview

Traditional culturing methods have shown that survival characteristics of some viable bacteria are not always detected.  (Ashbolt et al)

The surviving bacteria have been viable but non-culturable(VNC) They are also called somnicells. (Ashbolt et al) 

The growth rate of these bacteria, which are often pathogenic is so slow yet they retain their ability to cause disease.  This characteristic of pathogenic bacteria has been a problem for health authorities in devising regimes for testing water quality.  In USA, an outbreak of hepatitis A was caused when the victims ate raw oysters from waters that complied with bacteriological standards.  Unfortunately, the testing protocols did not apply to non-culturable bacteria. (Ashbolt et al)

VNC bacteria have a long stationary phase during which different genes are expressed compared to those in logarithmic phase or lag phases.                                               Index
 

Mycobacteria tuberculosis: an example of VNC bacteria

Mycobacterium tuberculosis is an example of a viable non-culturable bacterium. Mycobacterium is straight or slightly curved rods that are non-motile.  They sometimes exhibit branching. (Bergey) They form endospores in the stationary phase. (Ashbolt et al)  They are weekly gram positive and acid fast. (Starr et al)

Their slow rate of growth, doubling times from 18-24 hours, and isolates taking from 4-6 weeks to culture depending on method used, is due to environmental factors, but is ultimately the metabolism of the organism. (McMurray)

M.tuberculosis is a facultive parasite and can live intra and intercellularly. (Brock et al) It is the cause of the tuberculosis infection in humans and has severely affected persons infected with the human immunodeficiency virus (HIV) (McMurray)                                                        Index
 

Cell Structure

The cell walls of M.tuberculosis contain peptoglycan that is covalently bound to an arabinose-galactose-mycolic acid polymer. (Brock et al)  The lipid polysaccharide peptoglycan confers hydrophobic character. (Brock et al)

Acute infections, multiply in the extra cellular fluid, and chronic infections live intra cellularly. (Cole et al) The seriousness of the chronic infection is because whilst living within the cell, it is protected from the immune system of the host (Brock) and any drug therapies they may take.(Lumb)  People with chronic infections are carriers of the disease and so it is important to identify and treat them, preventing them spreading the disease. (McMurray                     Index
 

The Mycobacterium genome

The genome has 4,411,529 base pairs and contains about 4,000 genes in the circular genome.(Cole et al)  It has  a high guanine and cysteine residue (65.6%) which influences the amino acid content of the proteins.(Cole et al)

Much of its genome is devoted to coding for enzymes that are involved in lipogenesis and lipolysis. (Rosenkrands et al)  The high content of lipids in the cell walls makes it able to resist chemical agent like alkali and phenol for long periods. (Ashbolt)

The stationary phase or dormancy may be an indication of metabolic shutdown that has resulted from a cell-mediated immune response that can only contain but not eradicate the infection.  As either the host immunity diminishes through age or some form of immune suppression (i.e. AIDS), the dormant bacteria reactivate which will cause a symptomatic condition long after the original infection.

Although the metabolic basis of the dormancy is unknown, it is considered genetically programmed and involves intracellular signaling pathways. (Cole et al)                            Index

The genome has many insertion sequences and repetitive DNA with some regions having a higher than average percentage of G+Csequences. (Cole et al)

From 50 functional coding genes, there are three species of RNA molecules produced.  M.tuberculosis  has a single rRNA operon per genome(rrnAs) and two promoters, situated adjacent each other, one that was responsible for 80% of transcription. (Gonzalez-Merchand et al)

This was compared with a rapid growth M.smegmatis that has two operons (rrnAf & rrnBf) per genome.  One of the operons has three promoters and the other has only one. (Manganelli et al)

It is clear that the number of active promoters is fundamental to the transcription level of the cell.  In the case of M.tuberculosis, 80% of the transcription is being effected by one promoter, contributing to its slow growth and non-culturable status. (Manganelli et al)

The amount of RNA in the cell is related to the number of ribosomes, (Cole et al) which in turn correlates with the number of ribosomes per cell and the growth rate.(Manganelli et al)  RNA yield from M.smegmatis was nearly 3 times that of M.tuberculosis (Grunberg-Manango) further supporting the contention that the amount of RNA is related to the growth rate.            Index
 

Sigma Factors and Regulation of growth

Regulation of gene expression in response to environmental change is necessary for  survival of an organism.  Three heat shock sigma factors, sigB, sigE and sigH have been studied in M.tuberculosis. (Manganalli et al)  Disruption of the sigE sigma factor was found to make the mutant organism more susceptible to heat shock than the wild type. (Qi-Long Wu et al)  It was unable to tolerate oxidative compounds, SDS treatment and heat shock, but was not affected by acid pH stress.  (Manganalli et al)

The tolerance to pH stress may be derived from the mycolitic component of the cell wall (Newton et al) and may indicate that sigE is not involved in its transcription. (Manganelli et al)  Expression of the Lex A protein in Mycobacterium binds to a Cheo box motif, resembling the upstream region of  the SOS inducible genes in Bacillus subtilus. (Farahnaz et al) 

A probe for the sigma factor, RpoS, essential in the expression of stationary induced genes has been suggested as a means of detecting VNC bacteria. (Manganelli et al)  This would enable diagnosis of the disease state and prescription of therapeutic drugs more quickly than awaiting a positive culture.                                                                                                            

A correlation has been found between physiological activity and the number of ribosomes in the cell.  The level of transcription is also controlled by the number of ribosomes available to translate the transcript. (Grunberg-Manago)   Whilst any number of promoters and initiators may produce mRNA, if that cannot be translated into a viable protein, then cell metabolism will not increase. (Gonzalez-Merchand et al)                                                                             Index
 

Metabolism of M.tuberculosis

M.tuberculosis is able to metabolise various carbohydrate, hydrocarbon, ketone, carboxylic acid and alcohols as well as operate under both aerobic (using citric acid cycle) and anaerobicaly (using phosphorylative electron transport). (Brock)  This ability to adapt to its environment adds to its pathogenicity as it can either compete with the lung for oxygen or survive in the anaerobic/microaerophilic environment of the granuloma (nodule formed at the site of persistent infection or inflammation. (Martin et al)

M.tuberculosis also has 250 different enzymes involved in the metabolism of fatty acids, which are 5 times as many as is found in E.coli. (King et al) 

With the complete genome of M.tuberculosis now available, control of the disease state is centering on immunization as the only viable solution.  DNA vaccination in various forms, exploiting the knowledge of the genome has become a focus of research. (WHO info page)                                                                                                                           Index
 

Interesting sites related to Mycobacterium tuberculosis

Note: You Must Use Your Browsers 'Back' Button to Return to This Site After Using the Links Below

World Health Organisation (WHO) Tuberculosis Information Page

Images of Mycobacterium tuberculosis 1

Images of Mycobacterium tuberculosis 2

Mycobacteria tuberculosis Genome Map

                                                                                                                                    Index
References

Ashbolt, N (1995) Public Health Water Microbiology for the 21st Century.  Recent Advances in Microbiology (3) 1995

Brock, T.D., Madigan, M.T.(1991) Biology of Microorganisms(6ed)Prentice Hall

Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E. III., Techaria, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krough, A., McLean,J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M.A., Rajandream, M-A., Rogers, J., Rutter, S., Seeger,K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor, K., Whitehead, S., Barrell, B.G. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature (393) 537-544

Farahnaz, M., Colston, M.J., Davis, E.O. (1997) Characterisation of tuberculosis LexA: regognition of a Cheo (Baccillus-type SOS) box. Microbiology V143 N3 929-938.

Gonzalez-Merchand, J.A., Colston, M.J., Cox, R.A. (1998) Roles of multiple promoters in transcription of ribosomal DNA: effect of growth conditions on precursor rRNA synthesis in Mycobacteria. Journal of Bacteriology Nov. 5756-5761.                                             Index

Grunberg-Manago, M. (1999) Messenger RNA stability and its role in control of gene expression in bacteria and phages. Ann Rev Gen 193-199

Haverkort, F (1994) The ‘Atypical’ Mycobacteria and Human Disease. Recent Advances in Microbiology (2) 1994

Lumb, R. (1993) The current status of Tuberculosis.  Recent developments in the laboratory diagnosis of Mycobacterial disease. Recent Advances in Microbiology (1) 1993

Manganelli, R., Voskuil, M.I., Schoolnik, G.K., Smith, I. (2001) The Mycobacterium tuberculosis ECF sigma factor ?E role in global gene expression and survival in macrophages. Molecular Microbiology 41 (2), 423-437.

Martin, C., Timm, J., Rauzier, J., Rafael, G-L., Davies, J., Gicquel, B. (1990) Transposition of an antibiotic resistance element in Mycobacteria. Nature (345) 739-745

McMurray, D.N. (2001) Mycobacteria and Nocardia Medmicro Chapter 33 http://gsbs.utmb.edu/microbook/ch033.htm

Newton, S.M., Lau, C., Wright, C. W. (2000) A review of antimycobacterial natural products. Phytotherapy Research  V14 N5 303-322                                                                    Index

Qi-Long Wu, Kong D., Lam, K., Husson, R.N. (1997) A Mycobacterial extracytoplasmic function sigma factor involved in survival following stress. J. Bacteriology V179 N9 2922-2928

Sneath, P.H.A. (ed) (1986) Bergey’s Manual of Systematic Bacteriology Vol 2 Wiliams and Wilkins

Star, M.P. (ed) (1981) The Prokaryotes. A handbook on habits, isolation, and identification of bacteria Vol 2. Springer-Verlag 

Animated graphic satisfies conditions and is provided free by Animation Factory

Back to the top