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The heat, drugs and knockout systems ofMicrobial Biotechnology

Craig Daniels,1 Carmen Michán,2 Tino Krell,1

Amalia Roca3 and Juan L. Ramos1

1Consejo Superior de Investigaciones Científicas,Estación Experimental del Zaidín, Department ofEnvironmental Protection, C/ Prof. Albareda, 1, E-18008Granada, Spain.2Universidad de Córdoba, Campus de Rabanales,Department of Biochemistry and Molecular Biology,Edificio Severo Ochoa C-6, 2a Planta, 14071, Córdoba,Spain.3Bio-Iliberis R&D, Edificio BIC, lab. 203, Av. de laInnovación, 1, E-18100 Armilla – Granada, Spain.

The current issue of Microbial Biotechnology completesthe second volume of this journal that aims to collect thebest fundamental science in the field of microbiologyrelated to biotechnological applications. The journal hasmaintained a regular flow of articles and has publishedmany extremely relevant articles in the field, some ofwhich are being extensively cited; an acknowledgementof their high scientific value.

In the upcoming issue of Microbial BiotechnologyMartín and colleagues (2009) provide a comprehensivereview of b-lactam production by deuteromycetes andfungi in their mini-review entitled ‘Regulation and com-partmentalization of b-lactam biosynthesis’. The authorsdiscuss in depth the biosynthetic steps involved in pro-duction of penicillins, cephalosporins, cephamycins andcephabacins. The processes of penicillin and cepha-losporin C biosynthesis are compartmentalized; begin-ning with the formation of a linear amino acid precursor(ACV), and the cyclization of this to isopenicillin N in thecytosol and proceeding to either penicillin G in peroxi-somes or cephalosporin C via penicillin N in peroxi-somes and the cytosol. Emphasis is placed on theimportance of peroxisomes (single membrane boundmicrobodies) as many of the significant biosyntheticsteps take place in these organelles. The compartmen-talization of the biosynthetic process allows division ofprecursors and enzymes and aids in the regulation ofthe reactions involved. Naturally, this also brings intoplay a number of important transport systems that arerequired to localize the enzymes and reaction interme-

diates during the biosynthetic process; many of theplayers forming these transport systems are still unde-fined. Recent exciting research on the transport mecha-nisms has led to the construction of strains able toproduce increased amounts of b-lactam (Nijland et al.,2008). The current discovery of the CefM efflux pumpprotein and its inactivation in an A. chrysogenum strainshowed the importance of precursor penicillin N trans-portation from the peroxisome during cephalosporin Cbiosynthesis (Teijeira et al., 2009). The authors con-clude that future experimentation on the methodologyof biosynthetic intermediate and enzyme transportbetween the cytosol and peroxisome, and transport ofthe final b-lactam products out of the cells will allowmanufacturers to manipulate the output of the final drugproducts.

Also in this upcoming edition the membrane fatty acidadaptation of anaerobic microorganisms is tackled in theprimary research article presented by Duldhardt and col-leagues (2009). They investigated the adaptation of bac-teria of the genus Thaurea, Geobacter and Desulfococcusto the presence of benzene, toluene, ethylbenzene,xylenes, chlorinated phenols and aliphatic alcohols. Theyreport that both T. aromatica and G. sulfurreducens havepredominantly palmitic and palmitoleic acids in their mem-branes but show an increase in the level of fatty acidsaturation in the presence of the tested compounds.While D. multivorans membranes were dominated bypalmitic and anteiso-branched fatty acids and the bacteriaresponded to treatment by increasing the ratio of straight-chain saturated fatty acids to anteiso-branched fattyacids. The authors also showed that the adaptiveresponses are reliant on de novo synthesis of fatty acidsand are therefore strictly correlated with cellular growth.So, although the anaerobic bacteria respond in a similarmanner to their aerobic counterparts (See Bernal et al.,2007) in the presence of toxic organic solvents the gen-erally reduced growth rate of anaerobes results in a muchdelayed adaptive response. These results and furtherresearch will undoubtedly be of great importance for thefuture use of anaerobic bacteria in the clean-up of oxygenstarved subsurface environments that have been con-taminated with toxic organic pollutants.

Microbial Biotechnology (2009) 2(6), 598–600 doi:10.1111/j.1751-7915.2009.00144.x

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd

Microcalorimetric techniques have an increasing impactin the fields of fundamental and applied biochemistry andmicrobiology. The use of different calorimetric approachesto study molecular interactions and protein unfolding inthe context of biotechnology was recently reviewed in thisjournal (Krell, 2008). Using these approaches, typicallysolutions of purified biomolecules are analysed. Thereview by Maskow and colleagues (2009) illustrates thepossibilities, advantages and limitations of a calorimetricanalysis of far more complex systems such as cultures ofactive microorganisms. The signal recorded in the latterapproach is thus not that of binding or thermal unfolding ofpure samples of macromolecules but the heat generatedby the metabolic activity of microbial cultures.

To this end a large variety of calorimetric instrumentswere developed, ranging from nanocalorimeters, able tofollow calorimetrically the metabolism of a single cell, tomegacalorimeters, which are microbial culture vessels inthe m3 volume range. The heat production rate as moni-tored by calorimetry can provide information on the bio-conversion stoichiometry and kinetics which are crucialparameters in the optimization of biotechnological pro-cesses. Several examples are presented which illustratethe gain in value of the information achieved by thecombination of respirometric data with calorimetric mea-surements. The biotechnological relevance of such mea-surements consists primarily in the real-time control ofbiotransformations. This is illustrated by the possibility todetect in real time the switch between oxidative and fer-mentative metabolism of S. cerevisae or the energeti-cally much smaller transition between meta and orthophenol assimilation pathways in bacteria. Such informa-tion can ultimately be used to control bioprocesses in away that maximizes the quantity of carbon flowing intothe desired product.

Heap bioleaching is currently the most successful tech-nology for the extraction of base metals from low-gradesulfide ores and scientific and commercial interest hasemerged to study the microbial ecology of industrialbioleaching processes (Diaby et al., 2007; Rawlings andJohnson, 2007; Garrido et al., 2008; Siezen and Wilson,2009). Remonsellez and colleagues (2009) describe thedynamics of an active microbial community in an industrialheap using molecular ecology/molecular biology tools.They propose that the chemical and physical conditions(such as pH and the Fe3+/Fe2+ ratio) are of the utmostimportance to determine which bacteria dominate com-mercial bioleaching processes. The authors reportedremarkable differences in the microbial communitiesdepending on the strip age. They identified a wide varietyof microorganisms that correspond to different phyloge-netic groups, although they are mainly bacterial speciesthat can reach up to 107 cfu ml-1 based on 16S rRNAquantification analysis. They were able to identify among

the ‘active’ microbes A. thiooxidans, Leptospirillumferriphilum, the Gram-positives Sulfobacillus spp. andAlicyclobacillus disulfidooxidans, and the archaeonFerroplasma acidiphilum.

An initial step in the industrial production of proteinsis the search for a suitable expression system.Microorganism-based methods are usually preferreddue to their ease in handling and their lower costs.The promoter-activator pair Pm/XylS combines a strongincrease in transcription after induction, with very lowbasal levels of expression, essential characteristics forfirst-class systems. Additionally, this duo provides severalother desirable characteristics: (i) the inducer does notneed a specific uptake mechanism, (ii) the intensity ofactivation can be easily modulated, and (iii) there aremany mutants available with altered specificities. In thisissue, Aune and collaborators present a method to con-struct mutant Pm/XylS expression systems with improvedyields (Aune et al., 2009). The authors used a systematicapproach that involved directed evolution with error-pronepolymerases on the xylS coding region together with afusion of Pm to the b-lactamase gene; alterations in ampi-cillin resistance were detected in the presence of m-toluicacid. Using this methodology they obtained mutants witha phenotype of increased resistance to the antibioticunder inducing conditions, all with changes located in theNTD of the XylS protein, in agreement with several pre-vious reports that identified this region as responsible forinducer binding among other functions (Ramos et al.,1990; Michán et al., 1992; Ruiz and Ramos, 2002). Com-binations of mutations (identified in this paper or fromother authors) proved that incremental increases in tran-scription could be additional. Furthermore, the authorslooked for positive combinations by staggered extensionprocess (StEp), randomly combining xylS variants toobtain new chimeral proteins that could increase expres-sion under induced conditions almost 10-fold comparedwith the original system and still maintaining low unin-duced expression levels. Additionally, the results pre-sented offer useful information in the understanding ofhow XylS recognizes effectors and in the prediction of its3-D structure. The novel use of direct evolution followedby combination of the identified mutations provides aneasy tool to manipulate the induction characteristics ofregulators, particularly when their active sites are not pre-cisely confirmed.

Knowledge regarding genetic manipulation of non-model organisms should be improved in order todevelop new fields in microbial biotechnology. Traditionalapproaches were based on the construction of knockoutmutants, but this tactic is difficult to perform in severalorganisms including many filamentous fungi due to theiralmost zero rates of homologous recombination. Toovercome this difficulty, Kemppainen and Pardo (2009)

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© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 2, 598–600

report the construction of vectors (pSILBAs) for use ingene silencing in the mycorrhizal fungus Laccariabicolour; the system could potentially be used in otherbasidiomycetes. The authors constructed three differentsilencing cassettes with high stability, due to the use ofintrons as spacer sequences between the invertedrepeats in hpRNA. The plasmids obtained were fused tothe pHg Agrobacterium binary vector in order to incorpo-rate the ability for transgene integration. Constructionswere tested by silencing the Laccaria nitrate reductasegene and monitoring growth on nitrate; the strongestinhibition was obtained with pSILBAg that was designedto avoid inverted repeated promoter structures in thesilencing triggering cassette. Additionally, silencingstrength variations were observed among differentstrains carrying the same vector, a phenomenon fre-quently observed in RNA silencing studies. The authorsdemonstrated that, in this case, the inhibition levelsobtained were biased by the transcriptional activity of theintegration sites and not by the integration copy number.The new tools for gene disruption studies presented inthis work will certainly contribute to the knowledge ofgene functions in filamentous fungi, and therefore havemany future possibilities in biotechnological applications.

All of these exciting primary research articles andtheir potential applications form part of the relevantscientific literature that is being published in MicrobialBiotechnology.

References

Aune, T.E.V., Bakke, I., Drabløs, F., Lale, R., Brautaset, T.,and Valla, S. (2009) Directed evolution of the transcrip-tion factor xyls for development of improved expres-sion systems. Microb Biotechnol doi: 10.1111/j.1751-7915.2009.00126.x.

Bernal, P., Segura, A., and Ramos, J.L. (2007) Compren-satory role of the cis-trans isomerase and cardiolipinsysnthase in the membrane fluidity of Pseudomonas putidaDOT-T1E. Environ Microbiol 9: 1658–1664.

Diaby, N., Dold, B., Pfeifer, H-R., Holliger, C., Johnson, D.B.,and Hallberg, K.B. (2007) Microbial communities in a por-phyry copper tailings impoundment and their impact on thegeochemical dynamics of the mine waste. Environ Micro-biol 9: 298–307.

Duldhardt, I., Gaebel, J., Chrzanowski, L., Nijenhuis, I.,Härtig, C., Schauer, F., and Heipieper, H.J. (2009)Adaptation of anaerobically grown Thauera aromatica,Geobacter sulfurreducens and Desulfococcus multi-vorans to organic solvents on the level of membrane fattyacid composition. Microb Biotechnol doi:10.1111/j.1751-7915.2009.00124.x.

Garrido, P., González-Toril, E., García-Moyano, A., Moreno-Paz, M., Amils, R., and Parro, V. (2008) An oligonucleotideprokaryotic acidophile microarray (PAM): its validation andits use to monitor seasonal variations in extreme acidicenvironments with total environmental RNA. EnvironMicrobiol 10: 836–850.

Kemppainen, M.J., and Pardo, A.G. (2009) pHg/pSILBAgamma vector system for efficient gene silencing inhomobasidiomycetes: optimization of ihpRNA – triggeringin the micorrhizal fungus Laccaria bicolor. Microb Biotech-nol doi: 10.1111/j.1751-7915.2009.00122.x.

Krell, T. (2008) Microcalorimetry: a response to challenges inmodern biotechnology. Microb Biotechnol 1: 126–136.

Martín, J.F., Ullán, R.V., and García-Estrada, C. (2009) Regu-lation and compartmentalization of b-lactam biosynthesis.Microb Biotechnol doi:10.1111/j.1751-7915.2009.00123.x

Maskow, T., Kemp, R., Buchholz, F., Schubert, T., Kiesel, B.,and Harms, H. (2009) What heat is telling us about micro-bial conversions in nature and technology: from chip tomegacalorimetry. Microb Biotechnol doi:10.1111/j.1751-7915.2009.00121.x.

Michán, C., Zhou, L., Gallegos, M.T., Timmis, K.N., andRamos, J.L. (1992) Identification of critical amino-terminalregions of XylS. The positive regulator encoded by the TOLplasmid. J Biol Chem 267: 22897–22901.

Nijland, J.G., Kovalchuk, A., van den Berg, M.A., Bovenberg,R.A.L., and Driessen, A.J. (2008) Expression of the trans-porter encoded by the cefT gene of Acremonium chrysoge-num increases cephalosporin production in Penicilliumchrysogenum. Fungal Genet Biol 45: 1415–1421.

Ramos, J.L., Michan, C., Rojo, F., Dwyer, D., and Timmis, K.(1990) Signal-regulator interactions. Genetic analysis ofthe effector binding site of xylS, the benzoate-activatedpositive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon. J Mol Biol 211: 373–382.

Rawlings, D.E., and Johnson, B. (2007) The microbiology ofbiomining: development and optimization of mineral-oxidizing microbial consortia. Microbiology 153: 315–324.

Remonsellez, F., Galleguillos, F., Moreno-Paz, M., Parro, V.,Acosta, M., and Demergasso, C. (2009) Dynamic of activemicroorganisms inhabiting a bioleaching industrial heap oflow-grade copper sulfide ore monitored by real-time PCRand oligonucleotide prokaryotic acidophile microarray.Microb Biotechnol doi:10.1111/j.1751-7915.2009.00112.x.

Ruiz, R., and Ramos, J.L. (2002) Residues 137 and 153 atthe N terminus of the XylS protein influence the effectorprofile of this transcriptional regulator and the sigma factorused by RNA polymerase to stimulate transcription from itscognate promoter. J Biol Chem 277: 7282–7286.

Siezen, R.J., and Wilson, G. (2009) Bioleaching genomics.Microb Biotechnol 2: 297–303.

Teijeira, F., Ullán, R.V., Guerra, S.M., García-Estrada, C.,Vaca, I., Casqueiro, J., and Martín, J.F. (2009) The trans-porter CefM involved in translocation of biosyntheticintermediates is essential for cephalosporin production.Biochem J 418: 113–124.

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© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 2, 598–600