mycobacteria protocols

1 Mycobacteria Bugs and Bugbears Tanya Parish and Neil G. Stoker 1. Introduction Mycobacteria are gram-posmve, rod-shaped bacteria of the Actmomycete family, and therefore are most closely related to the nocardia, corynebacteria, and streptomyces. Their most characteristic feature is their complex cell enve- lope, containing a high percentage of lipids, which include the large-branched mycolic acids. This envelope makes the bacteria resistant to breakage and rela- tively impermeable to antibiotics, and is responstble for the acid-fast staining property used to identify the organisms. The genomic DNA contains a high guanme plus cytosine (GC) content, ranging from 58459% (I), which affects the utility of Escherzchza coli as a surrogate genetic host. Mycobacteria can infect most species of animals including rodents, birds, and fish. However, their importance lies m the fact that they include major human pathogens. Tuberculosis, caused by Mycobacterwm tuberdons, remains the most important mfectious cause of mortality in the world, and lep- rosy, caused by Mycobacterzum leprae, still afflicts large numbers of people. Other species can be pathogenic, of which perhaps the most concerning is Mycobacterium unum, which has recently become apparent as a major oppor- tunist m HIV-infected people in the developed world. Mycobacteria fall naturally and taxonomically into two main groups: slow- and fast-growers. The slow-growers include most of the major human and am- ma1 pathogens, whereas the fast-growers include nonpathogemc species, such as Mycobactenum smegmatis, which is widely-used as a convenient, if imper- fect, model organism. From Methods m Molecular Emlogy, Vol 101 Mycobacterra Protocols Edrted by T Parish and N G Stoker 0 Humana Press Inc , Totowa, NJ 2 Parish and Stoker 2. Molecular Biology: A Brief History The use of recombinant-DNA methods to study the molecular biology of the mycobacterta began m 1985, with the generation of genomlc-DNA libraries from M tuberculosis and hf. leprue (2-5). These were imtially used to identify genes encoding antigemc proteins by screenmg with serum or monoclonal antibodies (MAbs). E. coli IS a gram-negative organism with an average GC content of 50%, and it was soon realized that the majority of mycobacterial proteins were not expressed from then own promoters m this cloning host. Greater success was therefore obtained with expression libraries (6). The use of mycobacteria as hosts for recombinant molecules was made pos- sible by the development of cloning vectors based on mycobacteriophages and mycobacterial plasmtds. The tdentification of suitable antibiotic-resistance markers (mmally resistance to kanamycm), and the success of electroporation (see Chapter 11) to introduce such vectors into the cell, has led to the wide- spread use of nonpathogemc mycobacteria as clonmg hosts. The most popular has been M. smegmatu, because it is a fast-growing, nonpathogenic organism. Paradoxically, electroporation proved immediately possible with Mycobacte- rium bows BCG, whereas tt was necessary to isolate an electroporation-compe- tent mutant of M smegmatis (the most widely used bemg strain mc21 55) (7). Other mycobactena are now also being developed for use as clonmg hosts such as Myco- bacterzum vuccae (8,9). Most vectors are still based on pAL5000, a plasmid iso- lated from Mycobacterium fort&urn (10), although other replicons and integrating vectors have been used to a lesser extent (see Table 1). One of the main problems of the vectors for use m mycobacteria is the low- copy number (approx 2-5 copies per cell). This, coupled with the difficulty of lysmg mycobacteria, explains why most primary cloning steps are carried out in E. cob using shuttle vectors, whtch carry origins for both E colz and mycobacteria. A set of vectors are available for mycobacteria that can be transferred directly from E. colz by conjugation (see Chapter 10). This avoids the use of electroporation, which always carries the risk of creating aerosols by arcing. In addmon, the use of phage-based vector systems is gaming popularity because the efficiency of transformation is much higher than can be achieved for plas- mid-based vectors (see Chapter 12). The next obstacle to serious genetic analysis was the production of mutants. Some work with chemical mutagenesis has been carried out, although made more difficult by problems of clumping. Transposon mutagenesis has now been achieved both m M smegmatis and M. bows BCG, as has gene replacement, although much of the technology and many of the tools are still far from ideal. These are discussed m Subheading 3. Table 1 Useful Vectors for Mycobacteria Vector Type Based on Selection Features Reference pJRD2 15 Cosmid RSFlOlO KmalSmb Tropist4 Cosmid pAL5000 Km pus903 Integrating IS900 Km pMV361 Integrating L5 phage Km pEP3 Plasmid pNG2 HW pYT937 Plasmid pMSC262 Km pMB35 1 Plasmid pLR7 Km pSMT3 Plasmtd pAL5000 Wg pAUl51 Plasmid pAL5000 Hm pDE22 Plasmid pAL5000 Hyg pJAz6 Plasmid PAL5000 Sm pJAZl1 Plasnnd pAL5000 Km pCG63 Plasmtd pAL5000 Km pCG79 Plasnud pAL5000 Km&m pMYGFP 1 Plasmtd pAL5000 Km pMYGFPhsp60 Plasrmd PAL5000 Km pMRRO0 1 Plasmid pAL5000 Hgd pENlO Plasmid PAL5000 Hg pRCX3 Plasmtd pAL5000 Km pSUM series Plasmid pAL5000 Km pPE207 Plasmid pAL5000 Ame Broad host-range plasmtd Expression vector Compatible with pAL5000 based vectors Rephcon from a Mycobacterzum avzum plasmid Expression vector using hsp60 promoter Expression vector; targeted to membrane Expression vector to produce secreted protein Transfer by coqugation Temperature-sensitive orzw, transfer by coqugation, carries Tn6 11 Temperature-sensitive orzM Temperature-sensitive orzM carrying Tn611 Green-fluorescent protein (GFP) reporter hsp60-GFP fusion Mercury resistance Expression vector to produce extracellular proteins xyZE reporter gene Blue/white screening for clonmg origin of transfer from RSF 1010 (conJugative) (36) (37) (38) (39) (40) (41) (421 Chapter 2 1 Chapter 2 1 Chapter 2 1 (43) (43) (44) (23) (17) (17) (45) (46) (15) (47) (48) “Km-kanamycm resistance *Sm-streptomycin resistance ‘Hyg-hygromycm resistance dHg-mercury resistance eAm-apramycm resistance foorrM-ongm of rephcatlon m mycobactena Parish and Stoker 3. Molecular Techniques: General Problems Most of the problems associated with the use of molecular techniques to study mycobacteria can be related to one of the following problems. 3.1. Slow-Growth Rate The slow growers can take up to six wk to form colonies on a plate, and even the fast growers may take up to two wk. This can lead to problems with con- tammation of cultures, especially with fungi. Cycloheximide can be used to overcome the problem of fungal contammation somewhat. Most commonly used media have been developed for the isolation of mycobacteria from clmi- cal samples; although they contam partially-selective agents, such as malachite green, many require supplementation with nonautoclavable constituents, com- pounding the problem of contammation. Other media that do not require supple- mentation have been successfully used for fast growers (see Table 2). Media must be selected carefully, depending on the technique. The long incubation time can also lead to problems with plates drying out, and care must be taken to ensure that plates are well-wrapped m Parafilm or clmgfilm. 3.2. Clumping Owing to the nature of the cell wall, mycobacterlal cells tend to stick together when grown in liquid culture, and form macroscopic clumps even when grown with shaking. Some species are more prone to this than others. The clumping leads to problems because many standard techniques require dispersed cul- tures (e.g., optical-density measurement), or, ideally, single cells (for platmg, screening for mutants, and infection of tissue culture cells) The problem can be alleviated by the addition of detergents; Tween-80 is most commonly used. However, this may be only partially effective, and may not be possible where it is important not to alter the outermost layer of the cell wall, or where deter- gent-sensitive procedures such as phage infection are being used. Somcation can be used to break up clumps of mycobacteria, but for pathogenic species this must be carried out with a cup-horn sonicator (inside an appropriate safety cabinet) rather than with a probe, in order to contain the aerosols generated. Clumps may also be broken up by passing cell suspensions through a 23-gage needle several times, but this is unacceptable for pathogenic species owing to the risk of needle stick injuries and infection. 3.3. Resistance to Lysis Mycobacteria are particularly resistant to normal methods of chemical lysis owing to the nature of the cell wall. In addition, the wall contains large quanti- ties of polysaccharides, which can contaminate subsequent preparations of nucleic acids. This is discussed in more detail in Subheading 4. Mycobacteria 5 Table 2 Commonly used media for the growth of mycobacteria Liquid media Appropriate techniques Glycerol Alanine Salts Mtddlebrook 7H9 Broth0 Dubos Broth” Proskauer and Beck Sauton’s Lemco Broth Tryptrc Soy Brothb M9 Munmal Medium DNA preparation General General Surface-pellicle growth General defined medium General Phage mfectlon Protein preparation and NTG mutagenesis Solid media 7Hll agala 7H 10 agara Lemco agar Lowenstem Jensen (LJ) slopes M9 Mimmal Agar Tryptrc Soy Agar Top agar BCG top BCG agal” General General General Strain maintenance Protein purrficatron/auxotrophy screemng Phage mfectron Phage overlays Phage overlays for BCG Phage mfections for BCG aRequn-es supplementation 6No Tween. 3.4. Safefy Aspects One of the most important problems when working with pathogenic myco- bacteria is the need for containment (see Chapter 2). Any procedure that mvolves the generation of aerosols IS potentially dangerous and should be mmi- mized. Thus, the use of nonpathogenic species as model hosts for genetic stud- ies is of great rmportance and convenience. The lack of established, disabled host-vector systems means that relatively straightforward recombinant-DNA experiments using nonpathogenic hosts will have to be carried out m contain- ment facilities 3.5. Spontaneous Antibiotic Resistance As mentioned before mycobacterra can be broadly divided mto the slow- and fast-growing species. Most slow-growers possess only one ribosomal RNA (rDNA) operon; this unusual situatron means that resistance to agents such as kanamycm can easily be acquired by mutation in the rDNA operon itself (II), which is not likely to occur where there IS more than one operon. Therefore, 6 Parish and Stoker care must be taken when working with such anttbtottcs to mclude appropriate controls when attempting to introduce plasmids, and transformants should always be checked for the presence of the desired construct. Most of the fast growers contain two rDNA operons, and therefore have a much lower rate of spontaneous resistance to kanamycin. 4. Fractionation As mentioned in Subheading 3.3., mycobacteria are difficult organisms to lyse, mainly owing to the strength of the cell wall, and methods to isolate sub- cellular fractions have been developed to cope with this problem. The additton of glycme to growing cells can be used m order to weaken the cell wall before attempting lysis. Intact genomic DNA can be isolated relatively easily, and many methods-chemical, enzymatic and mechanical exist to achieve this (see Chapter 3). RNA isolation poses more problems, because mRNA is very un- stable with an extremely short half-life; therefore, lysis must be rapid m order to prevent degradation, and methods generally mechanical-have been de- veloped for this (see Chapter 6). The isolation of intact mRNA from mycobac- teria has allowed much more scope with respect to the study of gene regulation, and analyses of transcrtptton start-sites and transcriptional control of genes are now possible. Techniques such as differential display, RNA arbitrarily pnmed- polymerase cham reactton (RAP-PCR) and reverse transcrrption (RT)-PCR are now all being applied to these organisms (see Chapters 23 and 24). Such studies have the potential of dissecting out genes that are up-regulated in VIVO, and of examining the effect of phagocytosis on gene expression, both important topics in virulence. The preparation of protein fractions from mycobactena IS relatively straight- forward. Secreted proteins can be recovered simply from the medium supema- tant and subsequently concentrated. For cell wall-associated or cytoplasmic protems, the cells are generally broken open using mechanical methods and then centrtfuged to separate the sub-cellular fractions (see Chapter 7). Methods for the isolation and characterization of nonprotein cell wall components such as mycolic acids and lipoarabinomannan have been well-developed (see Chapter 8). 5. Genetic Tools 5.1. Vectors There is a general paucity of mycobacterral genetic tools, and the majority of plasmid vectors for use m mycobacteria have all been developed from one plasmid, pAL5000, originally isolated from A4 fort&urn (10). This plasmid has a low copy number m mycobacteria (less than five), so that preparation of large quantities of plasmid DNA from mycobacteria presents a problem. Rep- Mycobacferia 7 hcons derived from other natural plasmids have been used to a lesser extent (see Table 1). An addmonal problem is that these plasmids do not necessarily function in all mycobacterial species; for example, pAL5000-derived vectors function m most species including M. smegmatzs, but have been unable to trans- form Mycobactenum intracellular-e (12). Plasmid and cosmtd isolation has also presented a problem, owing to the low-copy number and the poor quality of DNA recovered. Therefore, most plasmid analyses have been conducted after transfer of plasmid DNA isolated from mycobacterta to E coli for analysis. Plasmrd and cosmid DNA can be successfully isolated from mycobacteria, but this requires an adaptation to the standard lysis methods (see Chapter 4). 5.2. Reporter Genes Several reporter genes have been used successfully in mycobacteria, mclud- mg those encoding P-galactosidase (13), chloramphenicol acetyl transferase (14), catechol2,3 dioxygenase (IS), luciferase (16) (see Chapter 19), and green- fluorescent protein (GFP; 17) ( see Chapter 20). These can be used to assay promoter activity and provide information about gene regulation and relative promoter strengths. 5.3. Expression Systems Mycobacterlal promoters do not function well in E. coli and many do not possess the standard consensus sequences. The situatron seems much more similar to Streptomyces, where several different classes of promoters occur (IS). There IS still a lack of well-characterized promoters for the expressron of heterologous proteins. The most widely-used promoters for expression m mycobacteria have been hsp60 and hsp70, which are constitutively expressed to a high level and can be further induced by heat shock. Targeting signals, such as the a antrgen-leader peptide or lipoprotein-attachment signal, can be used to dtrect the protein towards secretion or the cell wall, respectively (see Chapter 2 1). The use of inducible promoters, such as that of the acetamrdase of M smegmatis (I4), may be extremely useful in allowing controlled expression of genes. 5.4. Mutagenesis Chemical mutagenesis has been used for isolatton of mutants of several mycobacterral species (18-22) and has the advantage that spectahzed genetic vectors are not required, and that mutations, with effects ranging from total gene inactivation to subtle alteratrons of phenotype, may be isolated (see Chap- ter 13). However, it has several drsadvantages, including the use of dangerous chemicals, the mabihty to select cells carrying mutattons, the creation of unde- 8 Parish and Stoker fined mutants, the possibility of multtple mutattons, and difficulty in locating the defective gene. Transposon mutagenesrs has been developed for use m M smegmatis, BCG and M tuberculosis using different insertion sequences and delivery systems (23,2#). The apphcabrlity of a particular transposon to a mycobacterlal species depends on an effective delivery system, relatively random msertron, and the absence of the msertion sequence from the host strain. In M. smegmatis, a temperature-sensitive delivery system has been used m order to overcome the low-transposition frequency found with nonreplicating vectors (23) (see Chapter14); however, this has not been easy to adapt to pathogenic specres because of the narrow temperature range at which these species are viable. A BCG transposon-delivery system has been described using a partrally deficient plasmid orrgm, but the msertron sequence (IS) from whtch the transposon Tn5 was derived is present in M smegmatis, and consequently Tn5 cannot be used in this species (24). In addition, the transposition frequency wrth thus delivery system is low. An efficient phage-based delivery system has recently been suc- cessfully developed for M tuberculosis (25). 5.5. Recombination The creation of defined-gene knockouts using homologous recombmatron has not proved to be straightforward m mycobacterra. Although it has been achieved with relative ease in the fast-grower M. smegmatis (26-28) (see Chap- ters 15 and 16), nutial attempts m the slow-grower BCG were unsuccessful (27). This was partly owing to a reported high frequency of rllegitrmate recom- bination that resulted after transformation wrth a nonreplicating plasmrd. This situation was unexpected and is more similar to mammalian systems, where the frequency of illegitimate recombination is much hrgher than that of homologous recombination. This has frustrated attempts to create gene knock- outs in species such as BCG and M. tuberculoszs. Recently, homologous recombmation has been more successful, with both single crossovers and tar- geted gene replacement being achieved (29-32) (see Chapter 18). It seems that the combination of more laboratories attempting this procedure and the devel- opment of more efficient vectors will improve the situation m the future. Other slow growers such as certain strains of M intracellulare have been much more amenable to such studies (12) (see Chapter 17). 5.6. Genome Analysis The study of mycobacterlal genes ~111 benefit greatly from projects to se- quence the complete genomes of M tuberculosis, M. leprae, and M. avium. The sequence of M. tuberculosis H37Rv has been completed (49), and 1s avail- able in sequence databases (accession number AL123456) or from the Sanger Mycobacteria 9 Centre (http://www.sanger.ac.uk/), who are also completmg the M. leprae ge- nome sequence. A second stram ofM tuberculosis (CSU93) and M. avzum are being sequenced by the Institute for Genome Research (http://www.tigr.org/). The amount of information arising from these projects is immense, and an inte- grated database MycDB-is being used to organize the data (see Chapter 9). MycDB can be accessed on the World Wide Web at http://ww.biochem,kth.se/ MycDB The ready availability of sequence data for all A4. tuberculoszs genes 1s an exciting prospect and opens up many avenues for future study. The apph- cation of pulsed-field gel electrophoresis to mycobacteria 1s another welcome addition to the tools available for analysis of whole genomes (see Chapter 5). 6. Detection and Diagnosis The slow-growth rate of mycobacterla has meant that traditional tech- niques for diagnosing infection take a long time. Obviously, culture is a lengthy process, and subsequent drug susceptibility testing IS even more time consummg. The advance of molecular tehcniques has allowed the develop- ment of alternative technologies that have the advantages of sensitivity, specl- ficlty, and speed. Many PCR tests are being developed and have been evaluated usmg clinical samples (33); not only 1s PCR rapid, but it can be designed to identify mycobacterla at the species level (see Chapter 27). Other DNA-based tests have also been used. Epidemlologlcal studies have been greatly aided by the development of restriction fragment length polymor- phism (RFLP) typing (see Chapter 29), most commonly using the insertlon element ISdllO as a target, although other polymorphic elements have also been described (31,35). Spohgotyping provides another rapld means of not only identifying the species, but also typing strains as well (see Chapter 28). The addition of such techniques as rDNA sequencing adds to the versatility of speclatlon techniques (see Chapter 26). Biochemical technrques can be used in conJunctlon with molecular techniques to provide a high degree of confidence in asslgnmg species (see Chapter 25). Drug-susceptlbllity testing has also been Improved with the application of such techniques as polymerase chain reaction-single-strand conformatlon polymorphism (PCR-SSCP) to detect mutations m the genes coding for drug targets (see Chapter 30)and the use of the luclferase-phage system to assay for drug resistance phenotypes (see Chapter 3 1). Many other new and varied techniques are being developed and evaluated, and it is to be hoped that this will greatly improve both the detectlon and effective treatment of mycobacterlal diseases. 7. Conclusions The basic tools required for molecular analysis of mycobacteria are now available. They are still limited, but at least they provide the necessary founda- 70 Parish and Stoker tlons for future progress. The imminent completion of several genome sequences of both M. tuberculoszs and A4 leprae barely 12 yr after the first gene libraries were constructed is an extraordinary feat, and the challenge over the next 12 yr will be to improve the tools available m order to make the best use of the information now in our hands. References 1 Clark-Curtlss, J. E. (1990) Genome structure of mycobactena, m Molecular Bzol- ogy of the Mycobacterza (McFadden, J J , ed.), Academic Press Ltd, London, UK, pp 77-96 2 Clark-Curtlss, J. E., Jacobs, W. R., Docherty, M A , Rltchle, L R , and Cur&s III, R. (1985) Molecular analysis of DNA and construction of genomlc llbrarles of Mycobactersum leprae. J. Bacterlol 161, 1093-l 102. 3 Thole, J E R., Dauwerse, H. G., Das, P K., Groothms, D. G , Schouls, L. M., and van Embden, J. D A (1985) Cloning of Mycobactermm bow BCG DNA and expresslon of antigens m Escherzchza ~011. Infect Immun 50, 800-806 4 Young, R. A., Mehra, V., Sweetser, D , Buchanan, T., Clark-Curtiss, J , Davis, R W., and Bloom, B. R (1985) Genes for the maJor protein antigens of the leprosy parasite Mycobacterwm leprae. Nature 316,450-452 5 Young, R. A, Bloom, B R , Grosskmsky, C M , Ivanyl, J., Thomas, D., and Davis, R. W (1985) Dissection of Mycobacterzum tuberculoszs antigens using recombinant DNA Proc Nat1 Acad Scl USA 82,2583-2587. 6 Jacobs, W R , Docherty, M A, Curtlss III, R , and Clark-Curtlss, J. E. (1986) ExpressIon of Mycobacterium leprae genes from a Streptococcus mutans pro- moter m Esherlchla co11 K12 Proc Nat1 Acad Scz. USA 83, 1926-1930. 7. Snapper, S B., Melton, R E., Mustafa, S , Kieser, T., and Jacobs, W. R. (1990) Isolation and characterization of efficient plasmld transformation mutants of Mycobacterlum smegmatls Mol Mxroblol 4, 19 1 l-1 9 19 8 Garbe, T R , Barathl, J , Barnrm, S., Zhang, Y., Abouzeld, C , Tang, D , Mukherjee, R., and Young, D. B. (1994) Transformation ofmycobacterlal spe- cies using hygromycrn resistance as selectable marker Mzcrobzology 140, 133-138. 9 Houssaml-Iraqm, M., Clavel-Seres, S., Rastogl, N , and David, H L (1992) The expression of the Mycobacterlum aurum carotenogenesls operon is not repressed by the repressor of Mycobacterzum vaccae photoinducible carotenogenesls. FEMS Mw-obzol Lett. 99,233-236 10 Rauzler, J., Momz-Perelra, J., and Glcquel-Sanzey, B. (1988) Complete nucle- otlde sequence of pAL5000, a plasmld from Mycobacterium fortuztum. Gene 71, 315-321 11 Bottger, E. C (1994) Resistance to drugs targeting protein synthesis m mycobac- terra Trends Mw-oblol 2,416-421. 12 Marklund, B I , Speert, D P., and Stokes, R W. (1995) Gene replacement through homologous recombmatlon m Mycobacterzum intracellulare. J. Bacterrol 177, 61Ow105 [...]... exposed to the light Mycobacteria Methodology 2.6 Mycobacterium 17 gordonae This species is typical of scotochromogenic mycobacteria, which are commonly found in water and are often the cause of contammation in laboratory water supplies, in particular from the colonization of rubber tubing attached to laboratory taps The specieswas named after Dr Ruth Gordon, a pioneer of the taxonomy of mycobacteria and... conditions for future use 3 Cultivation of Mycobacteria Media for the cultivation of mycobacteria have mainly been developed for the isolation of M tuberculoszs from clmtcal specimens such as sputum, body fluids, and tissue Specimens from clmical sites are frequently contaminated with normal body flora and require exposure to strong acids or alkali m order to recover mycobacteria However, decontamination... tsolatmg DNA from gram-negative (18) and gram-positive (19,20) bacteria are not optimal for mycobacteria Several methods, wtth varying approaches to achieve effictent cell lysrs, are reported for the isolation of genomtc DNA from Mycobacterzumspp (Table 1) Protocols employmg mechanical or physical disruption of mycobacterial cells include homogenization with glass (7,21) or zirconium beads (22,231, rapid... similar to Mycobacterium tuberculosis The mycobacteria may be broadly divided into several groups: those that have not been cultivated m the laboratory (e.g., Mycobacterium leprae); those that are very difficult to cultivate (e.g., Mycobacterzum lepraemurzum); the slow-growmg species (e.g., 44, tuberculosis); and the more rapid growers (e.g., Mycobacterium fortuztum) 2 Mycobacterial Species Although there... Deretic, V (1994) Gene expression m mycobacteria transcriptional fusions based on xyIE and analysts of the promoter region of the response regulator mtrA from Mycobacterwn tuberculosis MoI Mxroblol 13, 1057-1064 16 Gordon, S , Parish, T , Roberts, I S., and Andrew, P W (1994) The appllcatton of luciferase as a reporter of environmental regulation of gene expression in mycobacteria Letters in Applied Mzcrobzology... incubation, with M smegmatis growing at up to 45°C and M phlei at 52OC 2 IO A vailability of Strains The majority of research on mycobacteria requires the use of well-recognized standard strains that are available from several National Collections The NCTC supply strains of mycobacteria by application to: The Curator, NCTC, Central Public Health Laboratory, 61 Colmdale Avenue, London NW9 5HT, 18 Allen... Stoker 12 27 Kalpana, G V , Bloom, B R , and Jacobs, W R (1991) Insertional mutagenesis and illegitimate recombmatton in mycobacteria Proc Natl Acad Scl USA 88, 5433-5437 28 Sander, P., Meier, A., and Bottger, E C (1995) RpsL+ A dominant selectable marker for gene replacement in mycobacteria Mol Mzcrobzol 16, 99 I-1000 29 Aldovmi, A, Husson, R N , and Young, R A (1993) The uraA locus and homologous... liquid and solid media may be made selective using either antibiotrcs or chemical agents The basic components of most mycobacterial-culture media originate from the work of Proskauer and Beck in 1894 (18) The medium they developed is still used by some reference collections to mamtam mycobacterial species 3.1 Egg-Based Culture Media The use of egg-based media was introduced by Dorset m 1903 (191, and... phlel + + + Mycobacteria Temperatureof growth “C 37 42 45 + + + f + f + + + + + + + + + + 52 + with an optimum temperature of 37°C are able to tolerate temperatures of 4045°C Certain fast-growing spectes are able to grow at even higher temperatures, e.g., A4 phlei at 52°C (see Table 1) 3.8 Storage and Maintenance of Cultures A variety of methods are available for the storage and maintenance of mycobacterial... scrofulaceum, some strains of the M avzum complex, M xenopl, M phlel, and M smegmatts Indeed, it is unlikely that any simple selective medium will be suitable for all species of mycobacteria 3.8.2 Long-Term Storage There are several ways of storing mycobacteria long term and the method of choice will depend on the intended laboratory application The methods most frequently used mvolve lyophilization by freeze-drying, . 1 Mycobacteria Bugs and Bugbears Tanya Parish and Neil G. Stoker 1. Introduction Mycobacteria are gram-posmve, rod-shaped bacteria of the. majority of mycobacterial proteins were not expressed from then own promoters m this cloning host. Greater success was therefore obtained with expression libraries (6). The use of mycobacteria. main problems of the vectors for use m mycobacteria is the low- copy number (approx 2-5 copies per cell). This, coupled with the difficulty of lysmg mycobacteria, explains why most primary