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Microbial Biotechnology from medicine to bacterialpopulation dynamics

Craig Daniels and Juan-Luis RamosEstación Experimental del Zaidín, Consejo Superior deInvestigaciones Científicas, C/ Prof. Albareda, 1,E-18008 Granada, Spain.

In recent issues of Microbial Biotechnology there havebeen several exceptional articles ranging in topic from theantagonism of biofilm formation in pathogenic bacteria,and prebiotic selection of positive bacterial fermentationsin the colon to the discovery of novel biotechnologicallyimportant enzymes encoded by bacteria of the deep-seafloor.

In January there appeared two stimulating articles inrelation to quorum sensing and antagonism of biofilmformation (Lee et al., 2009; Ueda et al., 2009). In theinitial publication Ueda and colleagues screened a trans-poson library of Pseudomonas aeruginosa PA14 in orderto discover which genes impact biofilm formation. Theyfound 137 mutants with enhanced biofilm capabilitiesand 88 with reduced biofilm formation. One of theirunique and interesting findings was that among themutants which showed dramatically decreased biofilmformation were seven mutants of the UMP syntheticpathway. Further biofilm analyses using compoundsfrom the UMP synthetic pathway showed that only uraciland not UMP or UTP was able to serve as an internalsignal for biofilm formation. Incredibly, transcriptomeanalysis of one of the UMP pathway mutants revealedthat all three known quorum-sensing systems wererepressed when UMP synthesis was inhibited. Thesefindings led the authors to screen currently availableuracil structural analogues for their influence on biofilmformation in P. aeruginosa; this led to the discovery that5-fluorouracil (a currently utilized anticancer drug) is apotent inhibitor of biofilm formation, which is able toabolish the quorum-sensing phenotypes and reducevirulence in an infection model while remaining non-toxicto P. aeruginosa. The discovery of these so called ‘anti-virulence molecules’ is of great importance to currentbiotechnology endeavours as these compounds are lesslikely to lead to developed resistance in bacteriabecause they do not affect bacterial growth (Cegelskiet al., 2008).

In the second article relating to quorum sensing andvirulence of P. aeruginosa Lee et al. show that indole and7-hydroxyindole (7HI) are able to reduce virulence.Indole is produced from L-tryptophan by many bacteriaincluding Escherichia coli and in fact is used by E. coli asa signalling molecule; influencing biofilm formation, motil-ity and acid resistance. Using DNA microarrays and phe-notype arrays the authors showed that both indole andhydroxylated indole have global affects on gene expres-sion in P. aeruginosa. The compounds decreased viru-lence factor production and swarming motility while alsoincreasing antibiotic resistance. Further testing in animalmodels confirmed that 7HI could reduce P. aeruginosacolonization and increase gastric clearance of the bacte-ria. As reported above for 5-fluorouracil these molecules(indole and 7HI) are non-toxic to the bacteria as they donot affect growth rate; but because they extensively altervirulence gene expression they have potential pharma-ceutical application in the treatment of P. aeruginosainfections.

In relation to the virulence topic the January issue ofMicrobial Biotechnology also contains a very pertinentreview by Anna Fàbrega and colleagues (2009) in whichthe mechanisms of action of quinolones are examinedand discussed from a clinical point of view. These drugsinhibit microbial DNA synthesis by targeting two essentialtopoisomerases, DNA gyrase and TopoIV; because DNAgyrase is present in prokaryotes and not in eukaryotes it isan excellent target for development of broad-spectrumantimicrobial compounds. Since the first use of nalidixicacid in 1962 four generations of more powerful quinoloneshave been developed and designed to overcomeacquired microbial quinolone resistance. The authorsemphasize that the mechanism of quinolone resistance isnot solely due to mutations in the target genes of thepathogens (gyrA, gyrB, parC and parE) but also from awide range of efflux pumps that efficiently prevent accu-mulation of the drug inside the cells. The authors showhow up to nine RND family efflux pumps can come intooperation to confer antibiotic resistance in the humanopportunistic pathogen P. aeruginosa. While efflux pumpsand target genes are often chromosomally encoded, theauthors also show that some plasmid-encoded proteins

Microbial Biotechnology (2009) 2(3), 304–307 doi:10.1111/j.1751-7915.2009.00110.x

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

can chemically modify the aromatic rings of quinolonespreventing the action of the drugs on their targets.Fàbrega and colleagues (2009) in this review analysedcase-by-case the mechanism of quinolone resistancein a wide range of human pathogens including severalenterobacteriaceae, the opportunistic P. aeruginosa, thenon-fermenting Acinetobacter braumanni, and Stenotro-phomonas maltophilia among the Gram-negative bacteriaand a number of Gram-positive pathogens such as Sta-phylococcus aureus and Streptococcus pneumonia. Anal-ysing and understanding these diverse mechanisms ofquinolone resistance will undoubtedly allow the futuredevelopment of broader spectrum high efficacy quinoloneantibiotics.

The functional food industry including the developmentand use of prebiotics is an emerging field in food science.In the January issue of Microbial Biotechnology Sánchezand colleagues (2009) report on the effects of prebioticoligosaccharides on the protein/carbohydrate fermenta-tion balance and microbial population dynamics of asimulated human intestinal microbial ecosystem. Basi-cally, prebiotics are food constituents that are not digest-ible in the upper gut and thus beneficially affect the hostby selectively stimulating growth and/or activity of one orof a number of health-promoting bacteria in the colon.Prebiotic treatments are potentially important for colonichealth in humans because they may lead to better fer-mentation conditions in the distal colon which is thesection with the highest levels of cancer. One of the exist-ing drawbacks of currently used prebiotic additives is thatthey are mostly fermented in the proximal regions of thegut and don’t survive long enough to reach the later distalcolon and rectal regions. The problem is believed to bemainly due to the small size and the lack of extensiveside-chains of the oligosaccharides used. The authorstherefore concentrate on Arabinoxylan-oligosaccharides(AXOS) which are a recently discovered class of candi-date prebiotics that are likely fermented in differentregions of the gastrointestinal tract due to their variedstructure. Sánchez et al. showed that different AXOSpreparations can have distinct impacts on the in vitrointestinal fermentation activity and microbial communitystructure of the digestive tract. Using AXOS with anaverage degree of polymerization (avDP) of 29 allowedthe oligosaccharide to reach the proximal colon and sus-tained supplementation with this AXOS decreased thelevels of the toxic proteolytic markers phenol and p-cresolwhile increasing the concentrations of beneficial short-chain fatty acids by 25–48%. Interestingly, the authorsshowed that the overall microbial communities were onlyslightly altered; an observation which suggests that thefermentation mechanisms within the colon were actuallybeing altered. The authors conclude that AXOS of differ-ent avDP can be fermented by intestinal bacteria leading

to positive human health effects and that they are there-fore promising candidates to modulate the microbialmetabolism in the distal colon.

Since the early days of molecular biology, proteinexpression has represented one of the ‘golden’ tech-niques in recombinant DNA technology. Regulated pro-moters are useful tools for many aspects related torecombinant gene expression in bacteria; including high-level expression of heterologous proteins and controlledexpression at physiological levels in metabolic engineer-ing applications. Svein Valla and colleagues (Brautasetet al., 2009) discuss the use of prokaryotic expressionsystems based on the AraC-XylS family of transcriptionalactivators. The systems function in a wide range ofmicroorganisms, including enterobacteria, corynebacte-ria, soil bacteria, lactic bacteria and streptomycetes. Priorto these applied uses, this family of regulators haveattracted scientific interest for decades as novel modelsystems for use in basic research on bacterial generegulation. This in turn has led to relevant informa-tion on the range of effectors recognized by these regu-lators so that their use to express heterologous genesis very well defined. Among the regulator/promoterpairs used are the XylS/Pm system of the TOL plasmidand AraC/PBAD, RhaR-RhaS/rhaBAD promoter, of E. coli.The discovery, characterization and implementation ofnew and flexible bacterial expression systems will likelybe invaluable for the future exploitation of newly discov-ered enzymes.

In relation to the search and discovery of new proteinfamilies in the March issue of Microbial BiotechnologySiezen and Wilson (2009) published a Genomics Updatearticle which presented an extensive review of the genom-ics of deep-sea and sub-seafloor microbes. In the articlethey emphasize the importance of this vast reservoirwhich is purported to contain > 1030 microbial cells(Whitman et al., 1998). The conclusion is that throughextensive surface sea-water samplings and metagenomicanalysis such as those conducted in The Sargasso Sea(Venter et al., 2004) and the Global Ocean Sampling(Yooseph et al., 2007) we have been able to discovermany new protein families; however, the numbers are stillrising and microbes from the deep-sea have yet to besignificantly exploited. Because deep-sea environmentsare characterized by low temperature (1–2°C), high pres-sure (1 MPa for every 100 m), high-salt and low-nutrientconditions microbes that live there differ greatly fromthose of shallow waters. For example, shallow watermicrobes do not require extensive chemotactic systems,flagella or pili because they live in a nutrient-rich high-oxygen environment with available sunlight; this is not thecase for deep-sea microbes. The authors also illustratethe current standing of the databases and computing toolsavailable for the cataloguing of the vast quantities of data

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that are becoming available (Siezen and Wilson, 2009).The coupling of extensive sampling techniques withmetagenomic strategies offers great promise for futureapplication of marine enzyme biotechnology to novel bio-catalyses using enzymes with high salt tolerance, thermo-stability and barophilicity.

A great complement to the genomics contribution bySiezen and Wilson (2009) is the mini-review by Ferrer andcolleagues (2009) in the preceding issue of MicrobialBiotechnology. In which several methodologies forharvesting of environmental DNA for metagenomics arepresented together with some sophisticated high-throughput screening tools that have been developed byAharoni’s lab. These well-refined techniques are based onin vitro compartmentalization approaches, which areuseful to search for novel activities or for rapid in vitroevolution of known enzymes. The focus of this mini-reviewis to demonstrate the power of small and large insertexpression library construction with lambda phage,cosmid and fosmid vectors; the vectors are implementedfor use in direct activity screening with the aim of discov-ering new enzymatic activities or novel variants of previ-ously established enzymes. This approach is in itself analternative to massive DNA sequencing of metagenomesthat has already revealed that over 60% of environmentalDNA represents novel sequences of unknown function.Ferrer and colleagues propose to go and fish for activitiesthrough the direct selection of activities based on the useof chromogenic or fluorogenic substrates that facilitatedirect selection, or indirectly through the use of genetictraps as those described by de Lorenzo (Garmendiaet al., 2008). Ferrer and colleagues detail and explain theminimal requirement for high-throughput screening andselection methodologies. A complementary approach tothe in vivo procedures is the in vitro compartmentaliza-tion (IVC) selection mode, also described in this mini-review. The IVC is based on water-in-oil emulsion, wherethe water phase is dispersed in the oil to form micro-scopic compartments. The system has been optimizedso that on average each droplet contains a single geneand serves as an artificial system for coupled transcrip-tion and translation, as well as detection of single mol-ecules. This technology, in addition, allows the rapidevolution of the enzymes to confer new properties to theproteins, such as enhanced thermo tolerance or widersubstrate specificity. The authors conclude that therational application of high-throughput technologies inmetagenome screening will unveil new enzymes that willbe valuable for innovative biocatalytic processes.

An interesting continuation of this line of research isthe potential use of the robust Pseudomonas putidaorganism; which is finding multiple applications in pro-duction of enzymes for biocatalytic processes. Therobustness of P. putida is derived from their ability to

react to multiple stresses such as those induced by thepresence of toxic molecules in the environment includinga complement of efflux pumps that prevent the accumu-lation of toxic molecules in the cell (Ramos et al., 2009).Second generation biofuel production is searching fornew molecules that can store higher energetic potentialthan ethanol. Among potential new biofuels are anumber of aromatic alcohols that can be produced bydiverting carbon flux from the biosynthesis of aromaticamino acids. Using a wide range of techniques Molina-Henares and colleagues (2009) have deciphered thebiosynthetic pathways and genes for the synthesis of thethree aromatic amino acids in this robust organism andhave therefore established the basis for the future engi-neering of this microorganism for the production of newbiofuels.

References

Brautaset, T., Lale, R., and Valla, S. (2009) Positively regu-lated bacterial expression systems. Microb Biotechnol 2:15–30.

Cegelski, L., Marshall, G.R., Eldridge, G.R., and Hultgren,S.J. (2008) The biology and future prospects of antiviru-lence therapies. Nat Rev Microbiol 6: 17–27.

Fàbrega, A., Madurga, S., Giralt, E., and Vila, J. (2009)Mechanism of action and of resistance to quinolones.Microb Biotechnol 2: 40–61.

Ferrer, M., Beloqui, A., Vieitez, J.M., Guazzaroni, M.E.,Berger, I., and Aharoni, A. (2009) Interplay of metagenom-ics and in vitro compartmentalization. Microb Biotechnol 2:31–39.

Garmendia, J., de las Heras, A., Calcagno Galvão, T., andde Lorenzo, V. (2008) Tracing explosives in soil with tran-scriptional regulators of Pseudomonas putida evolved forresponding to nitrotoluenes. Microb Biotechnol 1: 236–246.

Lee, J., Attila, C., Cirillo, S.L.G., Cirillo, J.D., and Wood, T.K.(2009) Indole and 7-hydroxyindole diminish Pseudomonasaeruginosa virulence. Microb Biotechnol 2: 75–90.

Molina-Henares, M.A., García-Salamanca, A., Molina-Henares, A.J., de la Torre, J., Herrera, M.C., Ramos, J.L.,and Duque, E. (2009) Functional analysis of aromaticbiosynthetic pathways in Pseudomonas putida KT2440.Microb Biotechnol 2: 91–100.

Ramos, J.L., Krell, T., Daniels, C., Segura, A., and Duque, E.(2009) Responses of Pseudomonas to small toxicmolecules by a mosaic of domains. Curr Opin Microbiol(in press).

Siezen, R.J., and Wilson, G. (2009) Genomics of deep-seaand sub-seafloor microbes. Microb Biotechnol 2: 157–163.

Sánchez, I., Marzorati, M., Grootaert, C., Baran, M., VanCraeyveld, V., Courtin, C.M., et al. (2009) Arabinoxylan-oligosaccharides (AXOS) affect the protein/carbohydratefermentation balance and microbial population dynamics ofthe simulator of human intestinal microbial ecosystem.Microb Biotechnol 2: 101–113.

Ueda, A., Attila, C., Whiteley, M., and Wood, T.K. (2009)

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Uracil influences quorum sensing and biofilm formation inPseudomonas aeruginosa and fluorouracil is an antago-nist. Microb Biotechnol 2: 62–74.

Venter, J.C., Remington, K., Heidelberg, J.F., Halpern, A.L.,Rusch, D., Eisen, J.A., et al. (2004) Environmental genomeshotgun sequencing of the Sargasso Sea. Science 304:66–74.

Whitman, W.B., Coleman, D.C., and Wiebe, W.J. (1998)Prokaryotes: the unseen majority. Proc Natl Acad Sci USA95: 6578–6583.

Yooseph, S., Sutton, G., Rusch, D.B., Halpern, A.L., William-son, S.J., Remington, K., et al. (2007) The Sorcerer IIGlobal Ocean Sampling expedition: expanding the uni-verse of protein families. PLoS Biol 5: e16.

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