Culture

The variety of nutritional requirements covering the whole spectrum of known bacteria is wide, and aspects of initial culture of bacteria that will be considered here will be restricted to those relating to the subsequent process of diverse preservation techniques. Reference should be made to standard textbooks for information on how best to grow different species, details of media formulations, pH, gaseous conditions and optimum incubation temperatures and times. However, a number of general factors must be borne in mind regarding culture for preservation.

Primary isolation

An obvious first requirement is to ensure that the culture is pure. Wherever practicable, the use of solid media is to be preferred to liquid media, since these allow plating out and subsequent single-colony isolations. In medical bacteriology, one plating out and single-colony isolation is usual for immediate investigations (for example, identification or determination of antibiotic sensitivity), where speed of obtaining an answer is the over-riding factor. For less urgent requirements and for preservation, however, it is advisable to go through two or even three successive platings out and single-colony isolations to ensure purity of the culture.

Enrichment

Primary isolation may sometimes be preceded by enrichment of the source material and usually this will be done in liquid media. Indeed, liquid media may be essential if the required bacterium requires, for example, good aeration or fluxing with special gases. Plating out from appropriate dilutions will yield single colonies and again, wherever practicable, further culture should be carried out on solid media.

The rapid advances taking place in biotechnology have introduced large numbers of scientists and engineers to the need for handling microorganisms, often for the first time. Questions are frequently raised concerning sources of cultures, location of strains with particular properties, requirements for handling the cultures, preservation and identification methods, regulations for shipping, or the deposit of strains for patent purposes. For those in industry, research institutes or universities with little experience in these areas, resolving such difficulties may seem overwhelming. The purpose of the World Federation for Culture Collections' (WFCC) series, Living Resources for Biotechnology, is to provide answers to these questions.

Living Resources for Biotechnology is a series of practical books that provide primary data and guides to sources for further information on matters relating to the location and use of different kinds of biological material of interest to biotechnologists. A deliberate decision was taken to produce separate volumes for each group of microorganism rather than a combined compendium, since our enquiries suggested that inexpensive specialised books would be of more general value than a larger volume containing information irrelevant to workers with interests in one particular type of organism. As a result each volume contains specialised information together with material on general matters (information centres, patents, consumer services, the international coordination of culture collection activities) that is common to each.

The WFCC is an international organisation concerned with the establishment of microbial resource centres and the promotion of their activities.

Introduction

Individual resource and information centres provide valuable services to biotechnology, but their role can be substantially enhanced if their activities are effectively co-ordinated. This has been recognised in the past, and a number of committees, federations and networks have been set up for this purpose at the national, regional and international levels. Although the origins and composition of existing organisations differ and their geographical locations are widespread, their common purpose is to support and develop the activities of resource and information centres for the benefit of microbiology.

International organisation

World Federation for Culture Collections

There are fewer difficulties in setting up national and regional co-ordinating mechanisms than international systems, and yet one of the first developments in this area was the formation of the World Federation for Culture Collections (WFCC). In 1962 at a Conference on Culture Collections held in Canada it was recommended that the International Association of Microbiological Societies (IAMS) set up a Section on Culture Collections. The Section was established in 1963. Five years later, at an International Conference on Culture Collections in Tokyo, the formation of the WFCC was proposed and an ad hoc committee, together with the Section on Culture Collections, drew up statutes which were agreed at a congress in 1970. Following the conversion of the IAMS to Union status, the WFCC is now a federation of the International Union of Microbiological Societies (IUMS) and an Organisation of resource centres 161 interdisciplinary Commission of the International Union of Biological Sciences (IUBS).

Introduction

Microbiologists are faced with consideration of exponential growth in their laboratories on a daily basis. As users of a chapter on information resources for biotechnology they are exposed to a double dose of exponential growth. First, the explosion of information technology itself is due to the massive amounts of computing power available at ever diminishing cost. In turn, a population of computer aware and computer literate microbiologists represent a growing demand for more sophisticated access to modern information technology. The community of information technologists in concert with microbiologists are responding to this demand with a multiplicity of initiatives using various strategies.

The resulting activity induces feelings of inadequacy in the authors of such chapters as this, since at the moment of delivery to the editors the information is out of date. Resources previously known only by rumour are tested. Simple facilities being tested as pilot projects are quickly made available to the community. Local data banks open their doors to regional and even world-wide participation. Databases on databases spring up because of the need to discover available resources. The net result is an ever increasing base of information resources for biotechnologists.

In some cases, useful resources fall by the wayside, as have at least two of the resources listed. They have been discontinued in the interval between the first and present versions of this chapter. The root cause of such discontinuing of effort is lack of appreciation by the initial funding bodies of the complexity and time scale involved in database initiatives of this sort.

Introduction

Bacteria are ubiquitous and although the most comprehensive compendium, Bergey's Manual, lists only two to three thousand named species, the diversity of habitats means that no single identification scheme can be devised for the whole spectrum. With bacteria, the species definition itself is pragmatic, since a species is that which, for practical purposes, we find useful to consider as a species. A listing of only two to three thousand species names – a small number compared with, say, fungi, or insects – perhaps tells us more about the restraint of bacterial taxonomists rather than about bacterial diversity. It is certain that a unit currently considered to be ‘species’ in one of those parts of the total bacterial spectrum that has come under much study (e.g. pathogens of the human gut; antibiotic producing soil organisms) does not correspond in taxonomic rank with a ‘species’ in a little-studied part of the spectrum.

Identification to species level, however broadly or narrowly defined, is a major part of bacteriological practical work. However, it may be important to know not only to what species the organisms belong, but whether two or more isolates are, in fact, the same strain. For example, from multiple isolates in a hospital, can it be deduced whether a particular strain is spreading?

Administration

Supply of cultures

The aim of service culture collections is to supply authenticated cultures to bona fide scientists, on request, promptly and without restriction on their ultimate use. The supply of certain bacteria such as pathogens (plant, animal or human) or patent strains will, however, be subject to statutory regulations and collections may impose further conditions. These may include, for example, proof that the requesting scientist holds the appropriate licence or permit to work with the cultures requested; that the request or order for cultures bears an authorised signature; and that an appropriate import licence is held. Supply of cultures to and from different countries, if pathogenic to man or animals, is subject to the International Air Transport Association (IATA) regulations whether sent by post or by air-freight. While the culture collection will effect the despatch of cultures as quickly as possible, delay can occur if those requesting the cultures are unaware of, or attempt to ignore, regulations. The collections, however, can only operate within the regulations.

Location of strains

The primary information about cultures available from a particular collection will be found in its printed catalogue. These, however, are not published as frequently as, say, catalogues of commercial suppliers of chemicals or equipment and so are never fully up-to-date. Many catalogues are now held on computers, and this enables quick and continual up-date by the collection and, in some cases, can be made available on-line (see Chapter 2).

Bacteria have a daily impact upon human activities. The emergence of the new biotechnology has increased the awareness by scientists of the long recognised need for reliable, permanent, culture collections which safe-keep viable exemplars of the many known bacterial species and varieties. There is now an increased awareness too that what is in fact conserved in service collections represents but a small part of the bacterial gene pool. Outside the recognised and long-established ‘service-supply culture collections’ there are many other centres whose holdings of cultures add to overall microbial, living resources available to scientists. There is an emphasis in this book on what defines a useful microbial resource: the cultures themselves, their documentation, and increasingly wide knowledge of their existence.

Today we are also in an age of developing information technology. Progress here enhances the existing resources, making it increasingly easy for individual scientists to access the great body of technical information associated with holdings of cultures. An additional benefit from the use of information technology to improve wider access to known information is to bring more clearly into focus gaps in our present knowledge and shortfalls in the presently conserved ranges of organisms available.

This book is an introduction to these resources, to culture collections, their holdings, and to the ways and means scientists responsible for their upkeep are exploiting information technology in the service of science. Hopefully, it will act as a stimulus to both research scientists and those engaged even in focused applied work. Reality dictates that often the distinction between research and applied science is blurred, but the extremes of each have need for authenticated, documented exemplars of the known microbial gene pool.

Studies of the physical properties of exopolysaccharides involve the application of a wide range of techniques; the interpretation of the results also requires a thorough knowledge of the chemical structure of the polymer. Electron microscopy has recently been applied to materials like xanthan to determine the persistence length of the molecule and to ascertain whether it is in a single- or double-stranded form. However, there are many limitations on interpreting the data thus obtained, owing to the possible introduction of artifacts both in the initial recovery of the polysaccharide and in sample preparation. Provided that the exact primary sequence and structure are known, X-ray fibre diffraction can supply information on polysaccharide conformation; circular dichroism provides a sensitive probe of the local environment of cation-binding sites.

Conformation

Determination of the molecular structure and conformation of a bacterial polysaccharide can be accomplished by X-ray diffraction of crystalline samples in the form of fibres. The techniques for orienting and crystallising the polymer use stress fields and annealing in the same way as they are applied to synthetic materials. The detail determined depends on the quality of the fibre diffraction pattern. The periodicity along the polysaccharide chain is visible in the approximately horizontal layer lines of the diffraction pattern. The spacing of the layer lines gives the pitch of the helical structure. Molecular model building can then be used, based on the known chemical repeating unit structure and standard values for bond angles and lengths and ring structures.

Polysaccharides are incorporated into foods to alter the rheological properties of the water present and thus change the texture of the product. Most of the polysaccharides used are employed because of their ability to thicken or to cause gel formation (Table 9.1). Advantage is also taken of the ability of some mixtures of polysaccharides to exhibit synergistic gelling: basically, for the two polymers to yield a gel at concentrations of each which will not in themselves form gels (Chapter 8). Associated with these readily measurable properties are others, such as ‘mouth feel’, which are more difficult to define but which also show some correlation with physical properties. ‘Mouth feel’ has been related to viscosity and, in particular, to non-Newtonian behaviour. This also relates to the masking effect of viscosity on the intensity of taste. There is also a specific relationship between the polysaccharide and flavours present in any food. Thus, corn starch and xanthan both provide good perception of sweetness and flavour when compared with gum guar or carboxymethylcelluose. In addition, polysaccharides are used because of their capacity to control the texture of foods and to prevent or reduce ice crystal formation in frozen foods; they may also influence the appearance and colour as well as the flavour of prepared foodstuffs (Table 9.2). It must also be remembered that many foodstuffs already naturally contain polysaccharides such as starch or pectin. Thus, addition of any further polysaccharide or polysaccharides will in all probability involve interactions with these compounds as well as with proteins, lipids and other food components.

Exopolysaccharides have a number of industrial applications which relate either directly or indirectly to medicine. One such is the use of dextran and its derivatives on both a laboratory and an industrial scale for the purification of compounds of medical interest, including Pharmaceuticals, and of enzymes for diagnostic purposes. Polysaccharides may also be used to encapsulate drugs for their gradual delivery and they may be used to immobilise enzymes employed for diagnosis or for the chemical modification of pharamaceutical products. These applications clearly utilise the functional properties of the polysaccharides, such as their rheology or capacity for gel formation. Alternatively, the pharmacological or other biological properties of the polymers may be employed. There are essentially three types of direct application of the biological properties of exopolysaccharides to medicine. The exopolysaccharides may be used as vaccines in preference to whole microbial cells or cultures. Thus side-effects due to other cell components such as lipopolysaccharides or proteins are avoided. On the other hand, not all polysaccharides are good immunogens, nor are the exopolysaccharides necessarily the major factors in the specific disease syndromes caused by the polysaccharide-producing microbial pathogens.

Several exopolysaccharides mimic eukaryotic polymers in their structural details. For this reason, they may be associated with certain specific diseases such as meningitis (in the case of sialic acid-synthesising bacteria). Some of these polysaccharides are, however, useful as substrates for determining enzyme specificity, and others are used as substitutes for the eukaryotic polymers. Finally, a number of exopolysaccharides having antitumour or antiviral activity have been identified.

Introduction

The biosynthesis of most exopolysaccharides closely resembles the process by which the bacterial wall polymers peptidoglycan and lipopolysaccharide are formed. Indeed, the three types of macromolecule share the characteristic of being formed of carbohydrates and associated monomers, being synthesised at the cell membrane and exported to final sites external to the cytoplasmic membrane. The only exceptions are the exopolysaccharide levans and dextrans, which are synthesised by a totally extracellular process and whose formation will be discussed later in this chapter.

Formation of the precursors for polysaccharide synthesis occurs within the cytoplasm. This is probably a necessity to ensure that they are thus readily available, as in many cases they are utilised for several different polymer-synthesising systems. As they are freely soluble in the cytoplasm, they can be readily channelled to the appropriate biosynthetic process occurring at or within the cytoplasmic membrane. Elucidation of the initial stages of polysaccharide synthesis has proved more difficult than was the case for polymers found in microbial and particularly bacterial walls. This has been mainly because of the lack of suitable selection systems for obtaining mutants and of antimicrobial agents specifically inhibiting polysaccharide biosynthesis. Even the preparation of cell-free systems or of membrane fragments is rendered more difficult by the presence of the viscous extracellular polysaccharides. Various cell-free systems, including membrane fragments from ultrasonically lysed cells, solvent-extracted cells or cells permeabilised with solvents or chelating agents, have been used.

Mutants have proved useful in studies where it has been possible to obtain microorganisms deficient in precursor synthesis (UDP-glucose pyrophosphorylase or UDP–galactose-4-epimerase, etc).

If one looks at the various areas in which microbial exopolysaccharides are currently employed, it may be possible to make some predictions about future usage. The increase in interest in the physical properties of these polymers, together with a much better understanding of the relationship between physical properties and chemical structure and the continued search for new polysaccharides, will inevitably lead to new discoveries. Relatively few of these are likely to have properties suited to new applications or their use in place of currently used polymers. There are two major constraints: legislative and financial.

In the food industry, xanthan is currently unique in its acceptability. As has been mentioned earlier, gellan from Pseudomonas elodea is currently undergoing safety evaluation. These two polymers can potentially fulfil many of the perceived needs of the food industry for microbial polysaccharides as well as replacing some established plant or algal products. Any new polymer would only have a small market niche and this would probably be insufficient to justify the expense of development and of the safety appraisal needed to obtain legislative approval. It is more likely that new applications will be found for the polysaccharides, such as xanthan, which already are approved. An exceptional situation exists in Japan, where the microbial polysaccharides, being regarded as natural products, are acceptable food ingredients. Perhaps some new polysaccharides will find applications in Japan, which may justify their introduction into other countries. One such might be curdlan.

In non-food applications, xanthan currently holds a commanding situation.

Introduction

While microbial exopolysaccharides, in common with similar polymers from other sources, are the substrates for degradative enzymes, the number of polysaccharases that have been isolated and characterised is relatively small. Only a small number of the polysaccharide-producing microbial species also yield enzymes degrading the same polymers. The exceptions include some of the bacterial species synthesising alginate and hyaluronic acid. A rich source of enzymes degrading bacterial exopolysaccharides has proved to be bacteriophages. These viral particles contain polysaccharases as part of the particle structure, usually in the form of small spikes attached to the base-plate of the phage. After phage infection, the bacterial lysates normally contain further amounts of the same enzyme in soluble form. The advantage of bacteriophages as sources of enzymes degrading polysaccharides is their freedom from other associated glycosidases, which might further degrade any oligosaccharide products. On the other hand, yields of phage-induced enzymes are low and they can only be regarded as laboratory tools of value in structural studies, unless the genes for the enzymes can be cloned and expressed on a large scale in microbial hosts. In addition, not all bacteriophages for exopolysaccharide-producing bacteria yield such enzymes.

There are very few commercially available enzymes acting on microbial polysaccharides. Consequently, the laboratory interested in using enzymes for structural determinations or for quality control must normally isolate its own enzymes. A number of polysaccharases have been obtained from bacterial and fungal sources by using enrichment procedures, with the polysaccharides as substrates.

Introduction

Although some of the earliest studies on bacterial transformation utilised as a model system polysaccharide production in Streptococcus pneumoniae and its relation to virulence, further progress in studying the genetics of polysaccharide synthesis has taken some considerable time. Much effort was applied to studies on the genetics of colanic acid synthesis in E. coli and Salmonella typhimurium, but this has proved to be a very complex system with numerous regulatory mechanisms. The complexity may perhaps be, at least in part, related to the relatively large hexasaccharide repeat unit of this exopolysaccharide and the ability of most bacteria synthesising it to produce more than one extracellular polysaccharide. Recently, however, detailed knowledge of the genetics of exopolysaccharide synthesis has derived from various systems. The interest in xanthan as an industrial product from X. campestris, and in the bacteria per se as plant pathogens, has prompted their study. Rhizobium species, as well as producing at least two different polysaccharides of potential industrial interest, have received attention because of their symbiotic relationship with leguminous plants and the associated bacterial fixation of dinitrogen. Alginate production by Pseudomonas aeruginosa has been studied because of the correlation between polysaccharide secretion and the infection of cystic fibrosis patients. Finally, a range of (mainly pathogenic) Gram-negative bacterial species have been examined, E. coli strains being used for a number of studies. All this information enables us to see some common aspects in the genetic control and regulation of exopolysaccharide synthesis; the concept of a ‘cassette’ of biosynthesis genes unique for each polysaccharide, first conceived in exopolysaccharide-synthesising E. coli by Boulnois and his colleagues, may well be at least partly applicable to many, if not all, exopolysaccharide-synthesising bacteria.

Introduction

When one considers that the number of microorganisms known to produce exopolysaccharides is very large indeed, the number of structures of these polymers which have been exhaustively studied is still relatively small. As with all polysaccharides, the microbial products can be divided into homopolysaccharides and heteropolysaccharides. Most of the former are neutral glucans; the majority of heteropolysaccharides appear to be polyanionic.

Three types of homopolysaccharide structure are found. Several are linear neutral molecules composed of a single linkage type. (Microorganisms do not appear to yield the ‘mixed linkage’ type of glucan found in cereal plants such as oats and barley.) There are also several polyanionic homopolymers and these, unlike the glucans, also contain acyl groups. Slightly more complex structures are the homopolysaccharides of the scleroglucan type, which possess tetrasaccharide repeating units due to the 1,6-α-D-glucosyl side-chains present on every third main chain residue. Finally, branched homopolysaccharide structures are found in dextrans.

Microbial heteropolysaccharides are almost all composed of repeating units varying in size from disaccharides to octasaccharides. These frequently contain one mole of a uronic acid, which is usually D-glucuronic acid. Very occasionally, two uronic acids are present. The uniformity of the repeat units is based on chemical studies and it is possible that some irregularities may be found, especially in the polymers composed of larger and more complex repeat units. The heteropolymers commonly possess short side-chains, which may vary from one to four sugars in length. Very rarely, the side-chains themselves may also be branched.