T I B T E C H - F E B R U A R Y 1989 [Vol. 7]

Biofilms at the interface between microbiology and

engineering The claim is continually made that biotechnology is not a discipline in its own right, but is an amalgam of genetics, microbiology, biochemistry, fermentation technology, process en- gineering and economics. But as each subject area advances and develops ever more sophisticated techniques, how often do we get the opportunity for interdisciplinary discussion in any depth not only of the operation and practical relevance of such technology, but also of the concepts and thinking behind it?

The balance was redressed to some degree by the recent Dahlem Work- shop* on the structure and function of biofilms. The Dahlem Workshops (this was the 46th in the Life Science series) are uniquely successful be- cause they are uniquely structured.

An organizing committee meets approximately one year in advance, when it defines the goal of the workshop, fixes the subject areas to be discussed by each of four groups, selects titles and authors for 16 background papers, and invites an interdisciplinary group of 48 scientists to take part in the work- shop. On this occasion the goal was 'To provide new concepts, experimental approaches and math- ematical models for the description and control of biofilms', and the subject areas for discussion were: ex- change processes at biofilm surfaces; spatial distribution of biotic and abiotic components i n the biofilm; cellular physiology and interactions of biofilm organisms; and physical and (bio)chemical processes in the biofilm matrix. The papers covered a complete spectrum from chemical

*46th Dahlem Life Science Workshop: Struc- ture and Function of Biofilms; 28 November - 2 December 1988, Berlin, FBG,

and physical analysis of biofilm components and processes, through mathematical and experimental modelling and specialist techniques, to a range of practical uses and consequences. The participants, from Europe, USA, Israel, Argentina, Australia and Japan, were roughly evenly divided between, on the one hand, microbiologists (molecular biology, cell physiology, ecology, medical and dental) and, on the other hand, environmental and process engineers, and physical and math- ematical scientists.

Prior to the meeting, the draft papers are circulated and the partici- pants asked to submit written com- ments and questions. Each paper is thus 'refereed' by all 48 participants, and these contributions, with the papers themselves, form the basis for the discussions at the workshop. It is important to stress however, that there are no presentations of papers or other material during the five days of the workshop itself. A further striking feature of the Dahlem meet- ings is that each group presents a written report of its discussions. These group reports are then pub- lished along with the background papers to give a permanent and valuable record, available to non- participants.

The meeting begins with each group deciding on its own detailed agenda, and these agendas (as with the group reports later) are then discussed by all participants in open session. The remainder of the time is taken up with discussipns in specialist groups. Only two groups meet at any one time; individuals from the other groups 'spectate' or contribute directly depending on the subject under discussion and their own experience and interests. This

ensures maximum interplay and cross fertilization amongst the groups and individuals, and the greatest chance for the workshop as a whole to achieve its goal. Group discussions concentrate not so much on what we know, as what we do not yet know, but need to know. Their deliberations generally focus on an effort to define specific needs that must be met for the next stages of advancement.

This particular workshop con- sidered biofilms from a number of independent viewpoints: (1) as ex- perimental systems capable of man- ipulation and analysis, where the approach may be physical (particle transport and attachment, hydro- dynamics and heat transfer, detach- ment) or biological (microsensors and molecular probes, physiology of attached organisms, influence of microenvironments); (2) as naturally occurring ecosystems (cyanobacterial mats, dental plaque, soil particles, groundwater systems); and (3) as biological systems of major practical and engineering significance (cor- rosion processes, colonization of medical implants, waste water treat- ment, hydrocarbon and xenobiotic biodegradation, immobilized cell re- actors).

A number of key features of biofilms were highlighted, generally with specific reference to the in- adequacy of our current knowledge:

(1) In many natural and man-made environments biofilms are the prin- cipal sites of biological activity. (2) Biofilms are heterogeneous, with discontinuities in both vertical and horizontal dimensions (relative to the substratum), involving organisms and biotic and abiotic components, creating physicochemical micro- environments. Methods of character- izing the spatial distributions remain relatively inadequate, but could be developed considerably [see (7)]. (3) Biofilms are dynamic structures: their heterogeneities vary with time. Even where a mature biofilm is at a steady state, the nature of that steady state is generally undefined in terms of growth, maintenance, sloughing and grazing by protozoa. (4) There is considerable uncertainty about how the processes of surface attachment and/or the interaction

@ 1989, Elsevier Science Publishers Ltd (UK) 0167 - 9430/89/$02.00

T IBTECH - FEBRUARY 1989 [Vol. 7]

with other organisms and the microenvironments characteristic of biofilms affect the phenotypes of organisms. (5) There is essentially no informa- tion on the dynamics of gene transfer between biofitm microorganisms; this could be of major practical significance in release to soil systems of genetically engineered micro- organisms. (6) Greater understanding is required of the various transport processes to and within biofilms, and the effects on these processes of variations in the proportions of base and surface film. (7) In determining the presence and spatial distribution of organisms within biofilms, there is need of im- proved culture techniques (based on low nutrient concentrations, gradient techniques, use of natural buffer

systems), and the further develop- ment of molecular probe technology and its application to such complex systems. New tissue-slicing tech- niques and scanning confocal mi- croscopy also have much to offer. (8) In both microbiological analysis and control of engineering processes, methods of continuous non-invasive in-situ monitoring (by microsensor or optical probe) would be invaluable. (9) The greatest stress was put on the value of experimental and mathematical modelling. This was seen as being necessary in order to bring structure and form to the study of such complex systems as biofilms, with the advantage of explicit assumptions, quantification and identification of areas of ignor- ance. Most importantly however, it became clear throughout the work-

shop that it is modelling which offers the best possible foundation for the interactive link between problems and solutions in bioengineering, and analysis and understanding in micro- biology.

The Dahlem model for a workshop of this type, and the subject matter of this particular meeting should be of wide interest to biotechnologists of several different academic persua- sions. Further information on the Dahlem Workshops can be obtained from: Dahlem Konferenzen, Tier- gartenstrasse 24-27, D-1000 Berlin (West) 30, FRG.

ALLAN H A M I L T O N

Department of Genetics and Micro- biology, Marischal College, Univer- sity of Aberdeen, Aberdeen AB9 1AS, UK.

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Antisense RNA makes good sense

In the past five years there have been increasing numbers of reports that expression of specific genes can be inhibited or downregulated by the presence of antisense RNA. Anti- sense RNA is complementary to messenger RNA. It is presumed to exert its effect at least partly by forming a stable base-paired structure with the mRNA, thereby preventing translation (although other mechanisms may also operate). Further, many cell types contain ribonucleases (e.g. ribonuclease N) which attack double-stranded RNA, and so the antisense RNA-mRNA base-paired structure may be de- graded. In bacteria, gene regulation by antisense RNA is a natural (although unusual) phenomenon. In eukaryotes, the presence of a par- ticular antisense RNA is achieved by insertion into the target organism of the gene's coding region in reverse orientation under control of a pro- moter in the normal orientation.

For plants, the inhibition of gene expression by antisense RNA was first reported in 19871 , and since then

there have been several further papers published on this topic. T w o 2'3 of these provide good ex- amples of the potential applications of the antisense RNA technique.

Chalcone synthase is a key enzyme in the biosynthesis of flavanoids. In Petunia there is a small multigene family of chalcone synthase genes 2, one of which is expressed in flowers, leading to the formation of pink and red pigments. A cDNA clone containing the whole coding se- quence of the floralty expressed gene was spliced in reverse orienta- tion between the cauliflower mosaic virus 35S RNA promoter (a strong constitutive promoter) and the termi- nation signal from the nopaline synthase gene of the Agrobacterium Ti plasmid. The recombinant gene construct was introduced together with a gene conferring resistance to kanamycin into leaf discs of Petunia and tobacco via an Agrobacterium binary vector transformation system and whole plants were grown up from the kanamycin-resistant cells.

In the transgenic plants of both

~) 1989, Elsevier Science Publishers Ltd iUK) 0167 9430/89/$02.00

species, there was some variation in the effect of the anti-chalcone- synthase gene. In some plants, the development of enzyme activity was extensively suppressed, no flava- noids were made in the flowers, and the flowers were white. At the other end of the scale, there were plants whose flavanoid content and hence pigmentation were indistinguishable from wild-type plants, and which exhibited only minimal inhibition of chalcone synthase activity. Between these two extremes were plants that had pale pink flowers or flowers with white and pink sectoring. These plants, as might be expected, showed intermediate levels of chalcone syn- thase activity.

The reason for this variation in the expression/effect of the anti- chalcone-synthase gene is not clear. As has been mentioned, the antisense gene was placed under the control of a strong Constitutive promoter, rather than the chalcone synthase gene's own promoter (which is presumably flower-specific). The group at the Free University of Amsterdam that carried out this work suggests that the constitutive promoter may be subject to 'position effect'. In other words, the site at which the foreign gene construct becomes inserted into the