Cell morphology

The cells of the genus Burkholderia correspond in their general characteristics to those of other aerobic pseudomonads: Gram-negative rods, straight or slightly curved, with rounded ends, usually motile when suspended in liquid. Motility is due to several polar flagella, but a single flagellum per cell has been reported for B. andropogonis, B. glathei, and B. norimbergensis.* The single flagellum of B. andropogonis is sheathed (Fuerst and Hayward, 1969a). One species, B. mallei, is nonmotile and lacks flagella (Redfearn et al., 1966).

Intracellular granules

The cells of species of the genus accumulate granules of carbon reserve material (poly-β-hydroxybutyrate, PHB, which may be part of a copolymer with poly-β-hydroxyvalerate, PHA). Proteins (“phasins”) have been found to be associated with the granules (Wieczorek et al., 1996). Only B. pseudomallei and some strains of B. mallei use extracellular PHB for growth (Ramsay et al., 1990). All species able to accumulate PHB can use the intracellular polymer when needed; however, in the description of some species, the statement “can use PHB” has been included, without indicating whether the PHB was endo- or extracellular.

The colonies of most species of the genus are smooth, but those of the human pathogen B. pseudomallei often have a rough surface.

Lipids

The first detailed analysis of the fatty acids of aerobic pseudomonads, using both saprophytic and phytopathogenic strains, demonstrated the possibility of establishing a correlation with the phylogenetic subdivision of the genus Pseudomonas, as classically defined, on the basis of rRNA–DNA hybridization (Oyaizu and Komagata, 1983). Years later, the results of this survey were confirmed and extended (Stead, 1992). The results of these investigations firmly established the fact that members of rRNA similarity group II of Palleroni et al. (1973), which includes the genus Burkholderia, have a fatty acid composition containing C_{14:0 3OH} and C_16:0, and C_{18:1 2OH}. Most strains also contained C_{16:0 2OH} and C_16:1. Even though hydroxylated fatty acids are present in the lipids of members of other groups, group II including Burkholderia is the only one having hydroxylated fatty acids of 14, 16, and 18 carbon atoms (Table 1). A recent evaluation of the taxonomic significance of fatty acid composition emphasizes the diagnostic value of the above results (Vancanneyt et al., 1996), confirming earlier findings on this approach for the characterization of major phylogenetic groups within the pseudomonads (Wollenweber and Rietschel, 1990).

Ornithine-containing lipids in Pseudomonas aeruginosa, P. putida, and B. cepacia represent from 2–15% of the total of extractable lipids. The amino acid was not found in the phospholipids that amount to more than 80% of all the extractable lipids (Kawai et al., 1988). An analysis of the polar lipids and fatty acids of B. cepacia has shown that the only significant phospholipids in this species are phosphatidyl-ethanolamine and bis(phosphatidyl)-glycerol. These characteristics, taken together with the unusual lipid profiles of B. cepacia, can be used as markers of chemotaxonomic importance (Cox and Wilkinson, 1989a).

A striking feature of the cellular composition of B. cepacia is the range of polar lipids, which include two forms (with and without 2-OH fatty acids) of phosphatidyl-ethanolamine and ornithine amide lipids. Variations in the lipid composition, as well as in pigmentation and flagellation, were observed as the consequence of changes in growth temperature and limiting oxygen, carbon, phosphorus, and magnesium supplies in the medium. Phosphorus limitation appears to be the only nutritional factor that results in a composition with polar lipids represented only by ornithine amide lipids (Taylor et al., 1998).

Interestingly, the 3-hydroxylated fatty acid of 10 carbon atoms is a component of the lipids of B. gladioli but not of those of B. cepacia, as indicated by lipid analysis performed on B. gladioli strains isolated from respiratory tract infections in cystic fibrosis patients (Christenson et al., 1989).

Differentiation of the plant pathogenic species of Burkholderia can be done by a direct colony thin-layer chromatographic method. Only minor uncertainties have been noticed with respect to the composition of aminolipids (Matsuyama, 1995).

The general qualitative profile of hydroxylated fatty acids indicated above is constant for a given species, although at least in one case (B. glumae) a subdivision of strains into two types is possible based on differences in composition. One of the subgroups, which included the type strain, had a composition that was similar to the bulk of rRNA similarity group II. The other was represented by strains that had significant amounts of the C_{10:0 3OH} fatty acid, and was unique in rRNA similarity group II in having the C_{12:0 3OH} fatty acid (Stead, 1992). For some of the components of the fatty acid profile, significant quantitative variations can be observed among the strains of different species of Burkholderia.

In contrast to the hydroxylated fatty acids, which have their origin in lipid A, the more abundant, nonhydroxylated fatty acids are mainly located in the cytoplasmic membrane. Their value as taxonomic markers is significant at the species level and less at the higher level of the RNA similarity groups. All strains of group II have C_16:0, C_{16:1 cis}, and C_{18:1 cis} nonhydroxylated fatty acids (Stead, 1992). The investigations of Komagata and his collaborators have established that Q-8 is the quinone characteristic of group II (Oyaizu and Komagata, 1983).

Hopanes have been detected in the composition of cells of B. cepacia (Rohmer et al., 1979), but the value of these compounds as chemotaxonomic markers is not known because later studies apparently did not include strains of other species of the group. This point perhaps warrants further attention.

Flagella

Motility can be observed in young cultures of strains of all species of Burkholderia, with the exception of B. mallei, which lacks flagella. Cells of the latter species do not even show twitching motility on the surface of solid media (Henrichsen, 1975a). Motility in liquid is due to one or, more commonly, several polar flagella. A single flagellum per cell has been reported for B. andropogonis, B. glathei, and B. norimbergensis (see descriptions in the list of species at the end of this chapter). The best-known example is that of B. andropogonis, whose single flagellum is sheathed (Fuerst and Hayward, 1969a) (Fig. 1).

SDS-polyacrylamide gel electrophoresis has been used for the characterization of the flagellins of different species of aerobic pseudomonads. Based on flagellin composition, B. cepacia strains have been divided into two groups. Group I has flagellin of molecular weight 31,000, whereas group II flagellin ranges from 44,000–46,000. Type I was serologically uniform, while group II was heterologous. The flagellin types of B. cepacia appear to be analogous to the two major flagellin types of P. aeruginosa, and they could be used as molecular epidemiological tools (Montie and Stover, 1983).

The methodology for the isolation of B. pseudomallei flagellin and its characterization has been described. Electrophoretic analysis under denaturing conditions results in monomer protein bands with an estimated M_r of 43,000 (Brett et al., 1994). O-polysaccharide-flagellin conjugates in this species have been described with respect to their structural and immunological characteristics (Brett and Woods, 1996).

Pili (fimbriae)

Peritrichous pili have been identified years ago in B. cepacia (Fuerst and Hayward, 1969b). They are thought to facilitate adherence to mucosal epithelial surfaces (Kuehn et al., 1992; Sajjan and Forstner, 1993). Twitching motility is correlated with the presence of polar fimbriae, a correlation that is supported by the fact that this type of motility is absent from B. cepacia, which has no polar fimbriae (Henrichsen, 1975a). No fimbriae have been observed in B. andropogonis (Fuerst and Hayward, 1969b).

One or more of five morphologically distinct classes of pili can be present in the cells of B. cepacia. Some of the types have been identified in cells of epidemically transmitted strains, and others in environmental isolates (Goldstein et al., 1995; Sajjan et al., 1995). The role of a 22-kDa pilin in binding B. cepacia to the mouth epithelial cells has been described (Sajjan and Forstner, 1993). Further details on fimbriae will be given in the section on pathogenesis for humans and animals.

Composition of cell envelope

The earliest data on the cell wall composition of B. cepacia were obtained in S. Wilkinson's laboratory, where it was found that the major components, myristic, 3-hydroxymyristic, and 3-hydroxypalmitic acids, were indications that members of this group had a different composition than other species of aerobic pseudomonads. These results are in agreement with those of fatty composition of whole cells (Samuels et al., 1973). The core polysaccharide contains glucose, rhamnose, and heptoses, but at most a very low phosphorus content. The lipopolysaccharide (LPS) has a low content of 3-deoxy-D-manno-2-octulosonic acid (KDO), and the side chain is basically a mannan. One added peculiar feature is the presence of an acid-labile amino sugar phosphate presumably associated with the lipid A.

The results have been confirmed, at least in part, by Manniello et al. (1979). Analyses of B. cepacia performed by these workers revealed the presence of rhamnose, glucose, heptose, and hexosamine, but no KDO, and the phosphorus content was found to be about one-third of that of P. aeruginosa. The presence of KDO in the LPS of B. cepacia, however, was later confirmed (Straus et al., 1990).

A method of extraction of LPS from B. pseudomallei has been described. As in the case with B. cepacia, the link between the inner core and lipid A is stable to acid hydrolysis (Kawahara et al., 1992). The LPS of clinical strains of this last species was composed of two polymers made of different repeating units, but both polymers contained D-rhamnose and D-galactose residues (Cerantola and Montrozier, 1997).

Five major outer membrane proteins have been isolated from several strains of B. pseudomallei, with M_r values ranging from 17,000–70,000. One of the proteins associated with the peptidoglycan acts as a porin through which small saccharides may diffuse (Gotoh et al., 1994b).

Outer membrane proteins of B. cepacia and B. pseudomallei that are inducible by phosphate starvation appear to be similar to the E. coli PhoE porin protein. The latter does not have the binding sites for anions and phosphate that are present in the analogous proteins from Pseudomonas species (Poole and Hancock, 1986).

One porin of B. cepacia is an oligomer composed of two proteins. The purified 81-kDa whole protein (OpcPO) upon heating gives a major 36-kDa protein (OpcP1) and a minor one (OpcP2) of 27 kDa. The association of these two components is noncovalent (Gotoh et al., 1994a). Recently, the major porin protein, (OpcP1), was partially sequenced, and the information was used for cloning the gene. Its sequence showed an open reading frame of a length in agreement with that of OpcP1 (Tsujimoto et al., 1997).

Additional information about porins and other components of the cell envelopes may be found in the sections on antibiotic susceptibility and antigenic structure.

Pigmentation

Pigmentation is by no means a universal character of Burkholderia. Some B. cepacia strains are not pigmented, whereas others produce phenazine pigments of a bewildering variety of colors when grown on solid chemically defined media containing different carbon sources. Pigmented strains of the species can be subdivided into two types on the basis of their pigmentation: some are yellow on glucose yeast extract peptone agar and others are various shades of brown, red, violet, and purple (Morris and Roberts, 1959). Morris and Roberts isolated the pigment from a purple-pigmented strain and demonstrated that its basic structure was that of a phenazine. In fact, two phenazine pigments—one yellow and the other purple—may be synthesized, both of which are water-soluble under neutral or alkaline conditions. A single strain can produce both types, only one type, or none. Because the pigments are soluble in water, both the colonies and the medium appear pigmented. Growth on King A medium2 often enhances pigment production.

In the author's experience, pigment production in Burkholderia is, as in many other cases, a striking but not very reliable taxonomic character, because pigment biosynthesis often requires conditions that cannot be precisely controlled.

Nutrition and growth conditions

The strains of Burkholderia species grow in media of minimal composition, without the addition of organic growth factors. Occasionally, strains are isolated from nature that grow extremely slowly but are stimulated by addition of complex organic mixtures such as yeast extract. Some of the fluorescent plant pathogens of the genus Pseudomonas fall in this category, and the phenomenon has also been observed for some B. caryophylli strains (Palleroni, 1984).

The ability to grow in media of very simple and chemically defined composition stimulated research on the nutritional versatility of the aerobic pseudomonads—among them, species later to be assigned to the genus Burkholderia. These studies have revealed a remarkable variety of organic compounds that can serve individually as carbon and energy sources for strains of some species (B. cepacia, B. pseudomallei). The types of compounds used for growth are the basis of the vast phenotypic information now available for many strains (Redfearn et al., 1966; Stanier et al., 1966; Ballard et al., 1970). One of the simplest chemically defined media used for nutritional studies is the one recommended for the hydrogen pseudomonads (Palleroni and Doudoroff, 1972).3

The nutritional investigations revealed that some strains of B. cepacia could utilize any of a list of 100 organic compounds (two-thirds of the list of tested substrates) (Stanier et al., 1966). Later work performed in various laboratories has enlarged the list considerably. This remarkable metabolic versatility of the species was unknown to plant pathologists, and its discovery rapidly converted B. cepacia into a fascinating subject for biochemical research. A sample of the nutritional properties of Burkholderia species is presented in Table 2, and some of these properties, together with general characteristics of taxonomic importance, are summarized in Table 3. Comparisons of nutritional properties of some related species are also to be found in later sections (Tables 5 and BXII.β.8). To the remarkable metabolic versatility of B. cepacia we have to add the ability to fix N₂ that is exhibited by some of the strains (Bevivino et al., 1994; Tabacchioni et al., 1995). This ability is also present in a closely related taxon, B. vietnamiensis (Gillis et al., 1995).

Table 2. Utilization of carbon compounds by some Burkholderia speciesa, b

• ^a For symbols see standard definitions; ±, slow. The + or − superscripts of the d symbol refer to the result obtained with the type strain, when known.
• ^b Data from Gillis et al. (1995), Viallard et al. (1998), and Palleroni (1984).
• ^c Carbon compounds used by all strains (with few exceptions; see Gillis et al., 1995) are fructose, fumarate, galactose, gluconate, glucose, mannose, glycerol, m-inositol, mannitol, sorbitol, acetate, dl-glycerate, dl-lactate, l-malate, pyruvate, succinate, d-α-alanine, l-α-alanine, dl-aminobutyrate, l-aspartate, l-glutamate, l-serine, l-proline, p-hydroxybenzoate. Carbon compounds not used by any strain: glycogen, methyl-mannoside, d-melezitose, inulin, starch, d-turanose, aesculin, creatine, 3-aminobenzoate, d-mandelate, phthalate, isophthalate.

Temperature for growth

Usually a temperature of 30°C is used for growth of all strains of the genus. Many of them, however, can grow well at 37°C and even at 40°C. Additional information on temperature relationships will be given in the section dealing with the description of individual species.

Oxygen relationships

All strains grow well under aerobic conditions. Some species (B. mallei, B. pseudomallei, B. caryophylli, B. plantarii, B. vandii) can also use nitrate as the terminal electron acceptor under anaerobic conditions. The original description of B. vietnamiensis (Gillis et al., 1995) indicates that the strains are able to reduce nitrate to nitrite, suggesting that this is the final stage of the reduction process. However, B. vietnamiensis is described elsewhere as a denitrifier. In view of this, the species was omitted from Table 4, which presents the general properties of denitrifying aerobic pseudomonads. In spite of this, denitrification was included among the general properties useful as differential characteristics for Burkholderia species.

Table 4. Characteristics useful for differentiation of denitrifying aerobic pseudomonadsa

• ^a For symbols see standard definitions. The denitrifying pseudomonads P. azotoformans, P. mucidolens, and P. nitroreducens have not been included in this table.

Metabolism and metabolic pathways

Knowledge of many areas of the general metabolism of species of the genus Burkholderia is scanty and fragmentary. The following refers to some aspects of the general metabolism of the genus, with the exclusion of the catabolism of aromatic compounds, which will be treated in a second part.

Extensive nutritional information is available on a large number of carbon substrates used individually as sole sources of carbon and energy for B. cepacia, B. gladioli, B. caryophylli, B. pseudomallei, and B. mallei (Redfearn et al., 1966; Stanier et al., 1966; Ballard et al., 1970; Palleroni and Holmes, 1981; Palleroni, 1984). In some of these reports, the information refers to names that are synonyms of some of the above (“Pseudomonas marginata” for B. gladioli, and “Pseudomonas multivorans” for some of the strains of B. cepacia). The information has been summarized in several tables in this chapter.

For B. vietnamiensis and B. andropogonis, a source of information on nutritional spectra useful for a comparison with other species of the genus, is given by Gillis et al. (1995). Less extensive surveys have been performed with B. glumae, B. vandii, and B. cocovenenans (Azegami et al., 1987; Urakami et al., 1994; Zhao et al., 1995).

As mentioned in the section on nutrition, some of the Burkholderia species are extremely versatile from the metabolic standpoint. This is particularly true of B. cepacia, the most versatile of all known aerobic pseudomonads, but B. pseudomallei and B. gladioli are similarly remarkable for their capacity of living at the expense of any of a long list of organic compounds as sole source of carbon and energy. Unfortunately, these species also have an infamous reputation as direct or opportunistic human and animal pathogens, and investigations on the saprophytic activities of some of the species are sparse because of the danger of handling the organisms in the laboratory.

All species of the genus Burkholderia that have been examined are able to accumulate PHB as a carbon reserve material, which they can degrade when nutrients in the medium become exhausted. However, use of exogenous PHB is an uncommon property among the aerobic pseudomonads. In Burkholderia, only B. pseudomallei and some strains of B. mallei are capable of degrading extracellular PHB (Redfearn et al., 1966; Stanier et al., 1966).

Table 5 summarizes data on arginine utilization and the occurrence of arginine deiminase in some representative members of the various rRNA similarity groups of aerobic pseudomonads. Interestingly, B. cepacia—a member of RNA group II that is notorious for its nutritional versatility and for the diversity of its catabolic pathways—can degrade arginine only through the use of the succinyl transferase pathway, although it can use 2-ketoarginine and agmatine, the products of arginine oxidase and arginine decarboxylase, respectively (Stalon and Mercenier, 1984; Vander Wauven and Stalon, 1985).

In a survey of lysine catabolic pathways in the pseudomonads, it has been reported that B. cepacia and P. aeruginosa use the pipecolate pathway and not the so-called oxygenase pathway. P. aeruginosa can also use the cadaverine pathway, but this is not operative in B. cepacia (Fothergill and Guest, 1977; Palleroni, 1984). These facts are summarized in Table 1 (BXII.γ.108, p. 334) of the genus Pseudomonas in Volume 2, Part B.

N₂ fixation has been detected in strains of B. cepacia isolated from plant rhizospheres (Bevivino et al., 1994; Tabacchioni et al., 1995). A different set of nitrogen fixing strains studied in another laboratory was found to be closely related to this species, and was assigned the new species name B. vietnamiensis (Gillis et al., 1995).

Some miscellaneous activities of interest of the lesser-known species of the genus include the following. An α-terpineol dehydratase—capable of converting the citrus compound limonene to α-terpineol—was partially solubilized from a particulate fraction obtained from B. gladioli cells (Cadwallader et al., 1992). A lipase from B. glumae has been purified and some of its properties have been described (Deveer et al., 1991; Cleasby et al., 1992). Cloning and sequencing of a lipase gene of B. cepacia (lipA) have been performed, and its expression is dependent on a second gene (limA) (Jorgensen et al., 1991).

In cells of B. caryophylli, a d-threo-aldolase dehydrogenase that catalyzes the oxidation of l-fucose and the “unnatural” sugars l-glucose, l-xylose, and d-arabinose, has been purified and characterized. It is inhibited by d-glucose and other natural aldoses (Sasajima and Sinskey, 1979). Some enzymatic activities of species other than B. cepacia may be of environmental importance. Thus, B. pseudomallei is capable of breaking the C-P bond in the utilization of the herbicide N-(phosphonomethyl)-glycine, which is known by the empirical name glyphosate. The genes controlling this activity have been cloned and sequenced (Peñaloza-Vazquez et al., 1995). Similarly, the phytopathogen and human opportunistic pathogen B. gladioli was found to be able to form and cleave C-P bonds (Nakashita et al., 1991; Nakashita and Seto, 1991). In a more general way, it has been suggested that B. gladioli probably participates in the degradation of some xenobiotic compounds in the environment (Cadwallader et al., 1992).

Several siderophores are produced by species of Burkholderia. In iron-deficient media, Pseudomonas aeruginosa, P. fluorescens, and B. cepacia synthesize salicylic acid, a compound of particular interest because of its siderophore capacity (Visca et al., 1993) and the fact that it is a precursor of another siderophore, pyochelin. Interestingly, salicylate is used as a source of carbon and energy by many strains of Burkholderia and of many other aerobic pseudomonads. An iron-binding compound produced by the great majority of B. cepacia strains isolated from the respiratory tract was named azurechelin and is capable of releasing Fe from transferrins (Sokol et al., 1992). Later, however, azurechelin was found to be salicylic acid (Visca et al., 1993).

Pyochelin production was found in half of 43 strains of B. cepacia isolated from cystic fibrosis patients. The siderophore has in its structure one molecule of salicylic acid and two molecules of cysteine (Cox et al., 1981). A siderophore of a linear hydroxamate/hydroxycarboxylate type, was discovered in B. cepacia and also found in B. vietnamiensis. It was named ornibactin and functions as a specific iron-transport system equivalent to that of the pyoverdin system of fluorescent pseudomonads. In the composition of ornibactins, there is a peptide, an amine, and acyl groups of different lengths (Stephan et al., 1993a, b).

In nature, these siderophores are often successful in competing for iron with siderophores of various other sources (Yang et al., 1993). Thus, pyochelin allows B. cepacia to grow in the presence of transferrin (Sokol, 1986).

In low Fe-content medium, B. cepacia excretes both pyochelin and a low molecular weight compound (1-hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone), which received the name of cepabactin. The structure resembles that of a cyclic hydroxamate, and it can also be considered a heterocyclic analogue of catechol (Meyer et al., 1989). The compound is related to synthetic hydroxy-pyridinones. Cepabactin had already been described as chelator (Winkler et al., 1986) and was known to have antibiotic properties (Itoh et al., 1979).

At least three different siderophores—ornibactins, pyochelin, and cepabactin—can be produced by a single B. cepacia strain. Other strains produce either two siderophores—ornibactins plus pyochelin or ornibactins plus cepabactin—and still other strains produce only pyochelin. In a survey of strains from a collection, 88% of the strains produced ornibactin, 50% pyochelin, and 14% cepabactin (J.M. Meyer, personal communication).

A recently identified member of the siderophore group produced by a strain of B. cepacia is cepaciachelin, a catecholate compound. The producing strain appears to be a unique example of a pseudomonad able to synthesize both hydroxamate and catecholate siderophores (Barelmann et al., 1996).

In summary, the variety of siderophores that B. cepacia is able to synthesize demonstrates once again the remarkable biochemical versatility of strains of this species.

All 84 strains of B. pseudomallei included in a study were found to produce a siderophore of approximately 1000 molecular weight. The compound, called malleobactin (Yang et al., 1991a), permitted cell growth in the presence of EDTA and of transferrin, and can trap iron from transferrin (at all pH values tested) and from lactoferrin. B. cepacia can use malleobactin as an iron-scavenging compound. However, pyochelin and azurechelin (salicylic acid) are more effective in trapping cell-derived iron as well as protein-bound iron (Yang et al., 1993).

Media with limiting phosphate concentrations enhance the production of a particular OM protein (OprP) of Pseudomonas aeruginosa. This protein is believed to be involved in phosphate transport and is only produced by species of Pseudomonas. Under similar conditions, both B. cepacia and B. pseudomallei produce proteins of a size similar to the PhoE protein of Escherichia coli and other enteric bacteria (Poole and Hancock, 1986).

Metabolism of aromatic and halogenated compounds

For many years this subject has attracted the attention of biochemists because of the striking ability of prokaryotes to degrade aromatic compounds that are resistant to chemical attack and are not readily metabolized by other organisms. In recent years, this interest has increased due to concern for chemically polluted environments and the obvious convenience of favoring these degradative activities as part of bioremediation strategies.

The first edition of this Manual included information on basic catabolic activities of the aerobic pseudomonads on simple aromatic compounds because of their obvious taxonomic implications (Palleroni, 1984). Much research has since been done on the catabolism of many aromatic compounds that include benzoate and derivatives, polycyclic aromatic hydrocarbons, biphenyl (particularly the halogenated derivatives), metabolic intermediates such as the halogenated catechols, and some important herbicides (24D and 245T). This field of research now contains a multitude of references, of which only a selected minority will be discussed here.

The investigations of Ornston and his collaborators on the biochemistry of degradation of aromatic compounds are of particular interest because the corresponding pathways were also analyzed for their phylogenetic implications. The β-ketoadipate pathway has taken center stage in these investigations. Natural selection has adopted many permutations in the distribution of its components, their regulation, and the genetic makeup, all of which have been highlighted in an excellent review (Harwood and Parales, 1996). Immunological cross-reaction was observed between the enzymes of one of the species (Pseudomonas putida) and the corresponding enzymes of other fluorescent Pseudomonas species. Cross-reaction, however, was also detected between the γ-carboxymuconolactone decarboxylases of P. putida and B. cepacia (Patel and Ornston, 1976). Although the results suggested that the interspecific transfer of the structural gene for the enzyme was not common among pseudomonads, it nevertheless seemed to have occurred between these two distantly related species. The cross-reaction did not extend to other enzymes of the two species, including the salicylate hydroxylases, which are structurally different, and for which an explanation based on convergent evolution has been proposed (Kim and Tu, 1989).

As mentioned in a description of the metabolism of para-hydroxybenzoate (POB), B. cepacia is able to convert meta-hydroxybenzoate (MOB) to gentisate by the action of a 6-hydroxylase (Yu et al., 1987). Interestingly, this finding relates to an earlier report on the formation of gentisate from MOB by Comamonas acidovorans, which is a member of the Betaproteobacteria in which Burkholderia is located (Wheelis et al., 1967). A description of the induction of the hydroxylase from B. cepacia has been published (Wang et al., 1987).

The genetic organization and sequence of genes of the α and β subunits of protocatechuate 3,4-dioxygenase of B. cepacia has been investigated, and there is extensive similarity to genes of other Pseudomonas species, although this similarity does not extend to the promoter sequences (Zylstra et al., 1989b). The pattern of induction of this and other enzymes of the POB metabolism has been examined (Zylstra et al., 1989a).

As a member of a microbial community, B. cepacia was the most competitive member among other aerobic pseudomonads in the degradation of toluene (Duetz et al., 1994). A novel pathway of toluene catabolism was described for this organism, with the participation of toluene monooxygenase, which is able to hydroxylate toluene, phenol, and cresol, and also to catalyze the degradation of trichloroethylene (TCE) (Shields et al., 1991). Cometabolism of TCE and toluene has been described (Landa et al., 1994). A constitutive strain was selected for the degradation of TCE (Shields and Reagin, 1992). Degradation of TCE by B. cepacia using the toluene monooxygenase system is expressed at higher capacity than the toluene dioxygenase systems present in other organisms (for instance, P. putida) (Leahy et al., 1996).

The toluene 2-monooxygenase from B. cepacia is a three-component system capable of oxidizing toluene to o-cresol and this to 3-methylcatechol. The catabolic features of this system resemble those of soluble methane monooxygenase from methanotrophic bacteria (Newman and Wackett, 1995). For the activity of toluene 2-monooxygenase on TCE, all its protein components and NADH are required. All protein components were modified during TCE oxidation, but reducing compounds such as cysteine protected the enzyme (Newman and Wackett, 1997).

Phthalate oxygenase is an enzyme specific for phthalate and closely related compounds. The system of B. cepacia, which requires the contribution of phthalate oxygenase reductase for efficient catalytic activity, is similar to other bacterial oxygenase systems. The B. cepacia enzyme can be isolated in large quantities and its stability is higher than that from other sources (Batie et al., 1987).

B. cepacia strains grow on fluorene and degrade this compound by a mechanism analogous to naphthalene catabolism. The system has wide specificity, and the range of substrates includes many other polycyclic aromatic compounds (Grifoll et al., 1995). But in spite of the remarkable catabolic versatility of the species, there are some limitations to the range of susceptible substrates. Thus, strains isolated on phenanthrene from polyaromatic hydrocarbon (PAH)-contaminated soils had limited capacity to use higher PAHs (Mueller et al., 1997).

For more than a decade, the degradation of the halogenated herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid (245T) by B. cepacia has been the object of attention by researchers, among them, A. Chakrabarty and his group (Haugland et al., 1990). In fact, B. cepacia grows luxuriantly on 245T. Although initially this species is not able to use phenoxyacetate, it can acquire this property by long selective pressure. Gene activation in the mutants seems to be due to translocation of insertion elements (Ghadi and Sangodkar, 1994). Polychlorinated phenols are produced in the degradation, and they are further metabolized by B. cepacia to the corresponding hydroquinones (Tomasi et al., 1995). The cloning, mapping, and expression of genes controlling the degradation by strains of this species have been under investigation (Sangodkar et al., 1988).

The study of spontaneous B. cepacia mutants unable to degrade 245T has permitted the identification of insertion sequences that facilitate or are required for growth at the expense of 245T (Haugland et al., 1991). The rapid evolution of the degradative pathway for this herbicide probably has been possible by insertion elements (such as IS1490) that play a central role in the transcription of 245T genes in B. cepacia (Hubner and Hendrickson, 1997). A 1477 bp sequence was repeated several times in a B. cepacia strain chromosome, and by the location it was concluded that genes involved in 245T degradation were actually recruited from foreign sources (Tomasek et al., 1989). Originally, foreign gene recruitment such as the mechanism postulated by Lessie and Gaffney (1986), may have been responsible for the acquisition of 245T degradation capacity. Better knowledge of the evolution of these pathways may facilitate the job of developing strains with enhanced degradative capacity (Daubaras et al., 1996a).

B. cepacia competes effectively in the microflora for the degradation of the herbicide 2,4-dichlorophenoxyacetic acid (24D) (Ka et al., 1994a). The enzyme that cleaves 3,5-dichlorocatechol in this pathway has been purified (Bhat et al., 1993). To monitor a strain in the degradation of 24D, a reporter gene system containing luxAB and lacZY was integrated into the chromosome of the strain, which thus could be readily identified in the community (Masson et al., 1993).

Oxygenase TftAB, capable of converting 245T to 2,4,5-chlorophenol, is an enzyme of wide specificity, since it can give phenolic derivatives of several other related compounds (Danganan et al., 1995). Two proteins, TftA1 and TftA2, characterized in the pathway, were sequenced and found to have similarity to BenA and BenB of the benzoate 1,2-dioxygenase system of Acinetobacter calcoaceticus, and to XylX and XylY from the equivalent system in Pseudomonas putida (Danganan et al., 1994). The B. cepacia enzyme that degrades 2,4,5-trichlorophenol, the intermediate in 245T catabolism, has been characterized and found to consist of two components (Xun, 1996).

1,2,4-trihydroxybenzene, another intermediate in 245T degradation, is a substrate for the enzyme hydroxyquinol 1,2-dioxygenase. The enzyme is specific, and it is a dimeric protein of 68 kDa (Daubaras et al., 1996b).

Environmental strains of B. cepacia are active in the degradation of polychlorinated biphenyls. As in other examples of aerobic metabolism of haloaromatic compounds, dehalogenation often occurs after ring cleavage (Arensdorf and Focht, 1995). In vitro constructed hybrids of B. cepacia could carry out total degradation of 2-Cl, 3-Cl, 2,4-dichloro-, and 3,5-dichlorobiphenyl (Havel and Reineke, 1991).

Chlorocatechols are key intermediates in the metabolism of haloaromatic compounds, and they are metabolized through reactions resembling those for catechol. A 2-halobenzoate 1,2-dioxygenase, a two-component system of B. cepacia strain 2CBS has a very broad specificity (Fetzner et al., 1992). However, in B. cepacia isolated from enrichment on 2-chlorobenzoate and in mutants blocked in different steps of the pathway, the 2,3-dioxygenase of the meta pathway predominated over the 1,2-dioxygenase (or ortho) system (Fetzner et al., 1989).

The dioxygenases and the cycloisomerases involved in the modified ortho pathways resemble the enzymes of pathways for nonhalogenated compounds, while the diene-lactone hydrolases needed in a later step are quite different (Schlomann et al., 1993). The results of work done on systems isolated from Alcaligenes and B. cepacia suggest that the hydrolases of the modified pathway may have been recruited from a different preexisting pathway, which was already operating before the start of industrial synthesis of halogenated compounds (Schlomann, 1994). When plasmid TOL is introduced into B. cepacia strains, these strains are able to grow with 3,4- and 3,5-dichlorotoluene, thus bypassing dead-end routes of chlorocatechols degradation (Brinkmann and Reineke, 1992).

The results of the experiments that have been briefly discussed in the preceding paragraphs often point to the high similarity among some components of peripheral metabolic pathways among organisms that are rather distantly related, suggesting horizontal transfer of the corresponding genetic determinants. One additional case is the high similarity between the genes coding for the cis-biphenyl dihydrodiol dehydrogenases of P. putida (gene bphB) and that of B. cepacia (Khan et al., 1997), and also between the bphC genes of both species, which code for the 2,3-dihydroxybiphenyl 1,2-dioxygenase (Khan et al., 1996b). On the other hand, in spite of having almost identical amino acid sequences, the biphenyl dioxygenase of B. cepacia (gene bphA1) and that of Pseudomonas pseudoalcaligenes have markedly different substrate ranges, the one for B. cepacia being wider (Kimura et al., 1997).

Additional details on the metabolism of aromatic compounds may be found in the section on plasmids.

Genetic characteristics

The genome of a strain of B. cepacia (ATCC 17616) consists of three replicons whose respective sizes are 3.4, 2.5, and 0.9 Mb, which, together with the 170-kb cryptic plasmid present in the strain, gives an overall estimate for genome size of approximately 7 Mb (Cheng and Lessie, 1994). The three large replicons had ribosomal RNA genes, as well as the insertion elements previously described by Lessie and collaborators. Studies on mutants and the associated reductions in size of the replicons provide a convenient framework for genetic analysis of this strain.

The genome of another strain of B. cepacia (ATCC 25416) was analyzed and found to contain four circular replicons of sizes 3.65, 3.17, 1.07, and 0.2 Mb (Rodley et al., 1995). The values total 8.1 Mb. Again the interpretation is that the genome is made of three chromosomes and a large plasmid, because of the presence of ribosomal RNA genes only in the three large replicons. An interesting additional observation is the fact that multiple chromosomes are not confined to B. cepacia but also are found in other members of rRNA similarity group II (B. glumae, Ralstonia pickettii, and R. solanacearum) (Rodley et al., 1995). A very useful review that highlights the remarkable genomic complexity and plasticity of B. cepacia has been published (Lessie et al., 1996).

A recA gene has been identified in B. cepacia that is able to complement a recA mutation in E. coli, also restoring UV and methylmethane sulfonate resistance and proficiency in recombination (Nakazawa et al., 1990). Additionally, an SOS box related to LexA-regulated promoters and −10 and −35 consensus sequences have been detected in B. cepacia. The predicted RecA protein sequence shows 72% similarity with that of P. aeruginosa.

Transposable elements that activate gene expression in B. cepacia have been identified by Lessie and his collaborators (Scordilis et al., 1987). This opened a new horizon on insertional activation and on the significance of a high frequency of genomic rearrangements that presumably are related to the remarkable versatility of the species (Gaffney and Lessie, 1987; Lessie et al., 1996).

In spite of all these developments, at present there is incomplete knowledge of gene expression and of regulatory mechanisms in species of Burkholderia because of the lack of an appropriate gene exchange system. In the absence of a conventional bacterial genetic system, some alternatives have been developed. One of the proposed methods of genetic analysis is based on transposon mutagenesis and complementation of mutations by means of cloned genes. In a particular application, a shuttle plasmid was constructed that could be used for cloning genes of B. cepacia involved in protease production (Abe et al., 1996).

Studies on population genetics have been carried out on a population of B. cepacia isolated from a southwestern stream in the United States, to examine the allelic variation in a group of loci using multilocus enzyme electrophoresis. The studies showed a low degree of association between the loci, or extensive genetic mixing. This evidence of frequent recombination (and consequent low levels of linkage disequilibrium) indicates that the structure of the population was not clonal (Wise et al., 1995).

The topology of a 23S rRNA phylogenetic gene tree agrees with the 16S rRNA tree (Höpfl et al., 1989).

Plasmids

A cryptic 170-kb plasmid that was discovered in B. cepacia ATCC 17616 has been the subject of much research. Derivatives of this strain carried versions of the plasmid containing various insertion sequences in different combinations. These elements, inserted in the broad host range plasmid RP1, served as probes to examine the extent of the reiteration of the various components in the genome of the organism. The results indicated a high frequency of genomic rearrangements, mainly the result of replicon fusions promoted by the insertion elements, that could help explain the remarkable biochemical versatility of the species (Barsomian and Lessie, 1986; Gaffney and Lessie, 1987).

Derivatives of a nonconjugative Pseudomonas plasmid (pVS1), carrying genes for mercury and sulfonamide resistance as well as segments required for stability and for mobilization by plasmid RP1, have been established in B. cepacia (Itoh et al., 1984). Some further constructions based on pVS1 could be used as cloning vectors (Itoh and Haas, 1985).

In many instances, the degradation of toxic compounds and environmental pollutants is controlled by genes located in plasmids. Some of them already have been mentioned, and only brief reference to some additional instances will be made here. A 50-kb plasmid is responsible for the degradation of para-nitrophenol by B. cepacia, following an oxidative route with the production of hydroquinone and nitrite. The plasmid can be conjugationally transferred (Prakash et al., 1996). A 70-kb plasmid in B. cepacia strain 2CBS carries a gene cluster with the determinant of an enzyme able to catalyze double hydroxylation of 2-halobenzoates with release of halogenide and CO₂ and producing catechol (Haak et al., 1995). A catabolic plasmid involved in the degradation of 4-methyl-o-phthalate was described and named MOP (Saint and Ribbons, 1990). Finally, a fragment of a plasmid involved in the catabolism of 4-methylphthalate in B. cepacia was sequenced and two open-reading frames were discovered, one of which encoded a permease that belongs to a group of symport proteins found in both pro- and eucaryotes. Information on this system could be used to improve the degradative capability for bioremediation purposes (Saint and Romas, 1996).

The novel pathway of toluene degradation mentioned in the section on metabolism of aromatic compounds is inducible and the corresponding genes are located in a plasmid. A strain of B. cepacia was found to carry two plasmids, one of 108 kb (named TOM) containing the genes for a toluene monooxygenase pathway that was expressed constitutively. The same strain also contained a small plasmid of less than 70 kb (Shields et al., 1995).

A new plasmid (pMAB1) with genes controlling the degradation of 24D in B. cepacia was characterized, and spontaneous negative mutants were isolated under nonselective conditions. Instead of the original 90-kb plasmid, these mutants had a smaller one (70-kb) or had lost it altogether. The activity could be regained by reintroducing the larger plasmid by electroporation. The 70-kb plasmid lacked a region that included the gene tfdC encoding the 3,5-dichlorocatechol 1,2-dioxygenase, whose sequence was identical with that of a well-characterized 24D degradative plasmid (pJP4) of Alcaligenes eutrophus. The similarity did not extend to the rest of the plasmids (Bhat et al., 1994). Another B. cepacia plasmid (pBS1502) was found to be able to control the early dehalogenation of 2,4-dichlorobenzoate (Zaitsev et al., 1991). There is also a report of the presence of a catabolic plasmid named MOP carrying genes involved in the catabolism of phthalate derivatives (Saint and Ribbons, 1990), and a small (2-kb) plasmid was implicated in the degradation of phenylcarbamate herbicides (Gaubier et al., 1992).

Plasmid analyses in combination with other typing techniques have been proposed for epidemiological studies on nosocomial infections by B. cepacia (Yamagishi et al., 1993). Early studies based on agarose gel electrophoresis of B. cepacia extracts demonstrated the presence of one or more plasmids in several strains of B. cepacia of plant and clinical origin. The plasmid composition, together with bacteriocin production and sensitivity, and pectolytic activity, could have applications in epidemiological studies (González and Vidaver, 1979).

Strains of B. cepacia from clinical and pharmaceutical origin carried large plasmids (146–222 kb) containing antibiotic resistance genes (Lennon and DeCicco, 1991). The nonconjugative B. cepacia plasmid pVS1 contained Hg and sulfonamide resistance genes, and a segment required for mobilization by RP1 (Itoh et al., 1984).

Bacteriophages

Most cultures of B. pseudomallei from collections have shown spontaneous phage production (Denisov and Kapliev, 1991). From the strains from a collection, 14 pure lines of bacteriophages belonging to two morphological types were isolated. The specificity of these phages was studied, and it was found that some strains of the host undergo poly-lysogeny, which was inferred from the fact that phages of different morphological types could be isolated from single strains (Denisov and Kapliev, 1995).

A generalized transducing phage was isolated from a lysogenic strain of B. cepacia, and half of more than 100 strains of the species were sensitive to it (Matsumoto et al., 1986).

Strain Berkeley 249 (ATCC 17616) of B. cepacia, which has been studied very intensively in Lessie's laboratory, carries an organic solvent-sensitive phage (Cihlar et al., 1978). Its sensitivity is attributed to alteration of a tail component provoked by the solvent. Results obtained by using other B. cepacia strains as hosts imply the occurrence of host restriction and modification systems. The phage has a head of 55 nm in diameter, a broad contractile tail of 15 × 145 nm, and double-stranded DNA of a molecular weight of about 3 × 10⁷.

Many years ago a bacteriophage lytic for a wide range of aerobic pseudomonads was isolated and tested against strains of different species, among which was “Pseudomonas multivorans” (later identified as a synonym of B. cepacia) (Kelln and Warren, 1971). B. cepacia was insensitive; the phage was lytic only for species of rRNA similarity group I (Pseudomonas sensu stricto) and not for members of other rRNA groups.

Bacteriocins

Early work performed on B. cepacia strains isolated from plants and from clinical specimens showed that the two groups could be differentiated by bacteriocin production patterns, onion maceration tests, and hydrolysis of pectate at low pH, thus suggesting the usefulness of these characteristics in epidemiological studies (González and Vidaver, 1979). Additional differences between strains from the two sources have been recorded (Bevivino et al., 1994) and will be discussed below (see Ecology, Habitats, and Niches).

A number of B. cepacia bacteriocins (“cepaciacins”) were defined in work done on a collection of 34 strains isolated from plant rhizospheres and human patients (Dodatko et al., 1989a). One of the cepaciacins consisted of protein and carbohydrate in a 3:1 molecular ratio. The bacteriocin was thermolabile, stable within a narrow range of pH values, and it was destroyed by proteolytic action. UV irradiation or mitomycin C stimulated its biosynthesis (Dodatko et al., 1989b).

A typing scheme has been described based on bacteriocin susceptibility and production by B. cepacia strains using six producer strains and a set of eight indicator strains (Govan and Harris, 1985). The majority of strains of a large collection were typed into a total of 44 combinations, and the typing scheme was found to be useful for the possibility of its application to epidemiological studies.

Antigenic structure

The LPS structure of different B. pseudomallei strains is quite homogeneous. Antibodies prepared with material from one strain react with all others (Pitt et al., 1992). In agreement with the degree of their phylogenetic relationships, cross-reactions are observed with B. mallei and, to a lesser extent, with B. cepacia.

Many features of the biological activity of LPS isolated from B. pseudomallei cells have been described, and the strong mitogenic activity that it has toward murine splenocytes has been attributed to unusual chemical structures in the inner core attached to lipid A (Matsuura et al., 1996). In addition, information is available about the identification, isolation, and purification of an exopolysaccharide of this species. As in the instance described above, the compound having a molecular mass of >150 kDa did not show cross-reactivity with any of the species of all the Pseudomonas rRNA similarity groups, with the exception of the closely related species B. mallei (Steinmetz et al., 1995).

Purification to homogeneity of flagellin from several B. pseudomallei strains gave monomer flagellin bands of M_r 43,400 Da. Passive immunization studies showed that a specific antiserum could protect animals from challenge by a B. pseudomallei strain of different origin (Brett et al., 1994).

Two monoclonal antibodies were found to be highly specific for B. pseudomallei when tested by indirect enzyme-linked immunosorbent assay and immunoblotting against whole-cell extracts of other Burkholderia species, fluorescent pseudomonads, and E. coli. One of the antibodies could agglutinate all 42 B. pseudomallei strains included in the study, thus providing a tool for rapid identification of the species using primary bacterial cultures from clinical specimens (Pongsunk et al., 1996).

Of the serological typing schemes devised for B. cepacia, the one most widely used is that proposed by Werneburg and Monteil (1989). Originally, the scheme described procedures for the preparation, adsorption, and titration of O and H rabbit sera, and it could define seven O (O1 to O7) and five H antigens (H1, H3, H5, H6, and H7) for the slide agglutination test and the agglutination and immobilization test, respectively (Heidt et al., 1983). The scheme later was supplemented with new serotypes, using strains of a different geographical origin, to make a total of 9 O and 7 H antigens (Werneburg and Monteil, 1989). Other immunological typing schemes have been proposed (Nakamura et al., 1986).

Wilkinson and his collaborators have studied the composition of the O-specific polymers from the LPS of B. cepacia strains belonging to groups O1 (Cox and Wilkinson, 1990b), O3, O5 (Cox and Wilkinson, 1989b), O7 (Cox and Wilkinson, 1990a), and O9 (Taylor et al., 1994a). The O9 group has repeating units that are also present in Serratia marcescens. In the same laboratory it has been discovered that the O antigen of the LPS of B. cepacia serotype E (O2) is composed of two different trisaccharide repeating units in a 2:1 ratio (Beynon et al., 1995), and that the same O specific polymer is found in the two related species B. cepacia and B. vietnamiensis.

Interestingly, the lipopolysaccharides extracted from B. cepacia and B. gladioli have a higher endotoxic activity and provoke a higher cytokine response than that from Pseudomonas aeruginosa (Shaw et al., 1995). This adds to the importance to the human pathogenic propensities of P. gladioli, an example of a plant pathogen of medical importance similar to that of B. cepacia. Plant-associated B. gladioli can be differentiated from other pathogenic and symbiotic bacterial species by a rapid slide agglutination test using polyclonal antisera conjugated to protein-rich Staphylococcus aureus whole cells (Lyons and Taylor, 1990).

Cross-reactivity of P. aeruginosa antipilin monoclonal antibodies with heterogeneous strains of P. aeruginosa and B. cepacia has been reported (Saiman et al., 1989).

Antibiotic susceptibility

For obvious reasons, most information on antibiotic susceptibility of species of the genus Burkholderia refers to only a few species of medical importance (B. pseudomallei, B. cepacia, B. gladioli).

All strains of B. cepacia that have been tested are sensitive to sulfonamides and novobiocin. Most are also sensitive to trimethoprim plus sulfamethoxazole, and to minocycline and chloramphenicol (Santos Ferreira et al., 1985). Both B. cepacia and B. gladioli are resistant to a wide variety of antibiotics (ticarcillin by itself or mixed with clavulanic acid; cefsulodin, imipenem, aminoglycosides, colistin, and fosfomycin) (Baxter et al., 1997). A catechol-containing monobactam (BMS-180680) was quite active (MIC90, 1 µg/ml; MIC90 is a minimum concentration which inhibits 90% of the strains tested) (Fung-Tomc et al., 1997).

Eighty percent of a collection of strains of B. cepacia was tested for inhibition by ceftazidime (Tabe and Igari, 1994). A number of quinolone analogs and derivatives (trovafloxacin, ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin, clinafloxacin, ceftazidime) were active on several Gram-negative, nonfermentative species, including B. cepacia. In comparison, the MIC for imipenem on B. cepacia and on Stenotrophomonas maltophilia was very high (Visalli et al., 1997).

In a comparison of the activity of many antibiotics against B. pseudomallei, a quinolone (tosufloxacine) and a tetracycline derivative (minocycline) appeared to be the most active (Yamamoto et al., 1990).

For years, the oral maintenance treatment of melioidosis has depended on the combined action of amoxicillin and the β-lactamase inhibitor clavulanic acid (Suputtamongkol et al., 1991). Biapenem, one of several carbapenem antibiotics tested against B. pseudomallei, was the most active against strains that showed a diminished susceptibility to third-generation cephalosporins (Smith et al., 1996).

Several resistance mechanisms operate in different species of Burkholderia. Resistance to cationic antibiotics in B. cepacia has been attributed to their ineffective binding to the outer membrane as a consequence of the low number of phosphate and carboxylate groups in the LPS, and the presence of protonated aminodeoxypentose (Cox and Wilkinson, 1991). The involvement of the outer membrane of B. cepacia in the resistance to polymyxin and aminoglycosides may also be related to a particular arrangement in the structure of the outer membrane, in which cation-binding sites on LPS are protected from polycations (Moore and Hancock, 1986).

An important factor in the antibiotic resistance in these organisms is porin permeability (Parr et al., 1987; Burns et al., 1996). However, a different interpretation is that resistance may not be a direct consequence of permeability of the porins, but instead may be related to the low number of porins per cell. A β-lactam-resistant mutant of B. cepacia and resistant strains of this species isolated from cystic fibrosis cases owe their resistance to a low porin content. These strains had reduced amounts of a 36-kDa outer membrane protein and did not express a 27-kDa outer membrane protein that can be a major porin or a major component of the porin complex of the cells (Aronoff, 1988). According to this view, the most common resistance mechanism in B. cepacia is the low porin-mediated outer membrane permeability, combined with multiple drug resistance due to an efflux pump system (Burns et al., 1996).

It may be of interest to mention here that growth in the presence of salicylate or other weak acids can induce resistance to antibiotics in B. cepacia, because these compounds have been found to be inhibitors of porin formation (Burns and Clark, 1992).

A number of β-lactams are susceptible to hydrolysis by a β-lactamase of B. cepacia, a metalloenzyme of type I that is induced by imipenem (Baxter and Lambert, 1994). A significant degree of similarity was found between the chromosomal β-lactamase of B. cepacia strain 249 and the enzymes of Pseudomonas aeruginosa and E. coli. Interestingly, in spite of differences in the mol% G + C content of the DNA of these organisms, the codon usage in B. cepacia resembled that of E. coli (Proenca et al., 1993).

The multiple resistance gene oprM of P. aeruginosa is part of a highly conserved efflux system. A gene homologous to oprM was identified in B. cepacia (Burns et al., 1996). Moreover, an intragenic probe hybridized the genomic DNA of several fluorescent pseudomonads and, in addition, the DNA of B. pseudomallei (Bianco et al., 1997). A lucid review is available on the participation of multidrug efflux pumps in the resistance of Gram-negative organisms to antibiotics (Nikaido, 1996).

The fusaric acid resistance gene of B. cepacia has been cloned and sequenced (Utsumi et al., 1991).

Antibiotic production

Antifungal antibiotics have been identified in strains of B. cepacia. Cepacidine A, composed of two related forms, cepacidine A1 and A2, is a cyclic peptide and xylose connected to a 5,7-dihydroxy-3,9-diaminooctadecanoic acid (Lee et al., 1994a; Lim et al., 1994). Two antibiotics previously discovered and described under the names cepacin A and B are not related to the cepacidines. The cepacins showed good antistaphylococcal activities (MICs of 0.2 and 0.05 µg/ml for cepacin A and B, respectively), but no significant activity against Gram-negative bacteria (Parker et al., 1984).

Another antifungal antibiotic is pyrrolnitrin (Jayaswal et al., 1993). The producing organism was named Pseudomonas pyrrocinia (Imanaka et al., 1965), now Burkholderia pyrrocinia. The antibiotic is also produced by B. cepacia and by some strains of Pseudomonas chlororaphis (Elander et al., 1968). A study by Burkhead et al. (1994) has examined the conditions of pyrrolnitrin by B. cepacia in culture and in the wounded areas of potatoes colonized by the organism.

Some compounds related to pyrrolnitrin (amino-pyrrolnitrin, and monochloroamino-pyrrolnitrine) also have antifungal properties (McLoughlin et al., 1992). The antibiotic activity of B. cepacia has been reported to antagonize the pathogenic activity of the sunflower wilt fungus (McLoughlin et al., 1992) and to be a suppressor of maize soil-borne disease (Hebbar et al., 1992). The influence of some environmental factors on the antagonism of B. cepacia toward Trichoderma viride has been analyzed (Upadhyay et al., 1991), as well as some morphological alterations and inhibition of conidiation of plant pathogenic fungi (Upadhyay and Jayaswal, 1992). A compound having both hemolytic activity and antifungal action was characterized and given the name cepalycin (Abe and Nakazawa, 1994).

Transposon mutagenesis could eliminate pyrrolnitrin production ability. However, the mutation failed to be complemented by the cloned gene because of difficulties encountered in mobilizing the carrier cosmids from E. coli to B. cepacia mutants (Jayaswal et al., 1992).

A group of eight cyclic peptides of antifungal activity, the xylocandins, has been isolated from B. cepacia and characterized (Bisacchi et al., 1987). A mixture of two of the forms (A1 and A2) showed potent anticandidal and antidermatophytic activities in vitro (Meyers et al., 1987).

The antagonistic activity of B. cepacia is not limited to antifungal activity. Of practical importance is the finding that metabolites produced by strains of this species can antagonize plant pathogenic agents such as Ralstonia solanacearum (Aoki et al., 1991).

Toxoflavin, an azapteridine antibiotic produced by B. cocovenenans, was identified more than 30 years ago (Lauquin et al., 1976). The production of toxoflavin is inhibited by the combined action of 2% NaCl and acidity (pH 4.5) in the medium (Buckle and Kartadarma, 1990). A monobactam antibiotic (MM 42842) is also produced by B. cocovenenans. It is related to a previously described antibiotic named sulfazecin. In addition, the B. cocovenenans strain synthesizes bulgecin, an antibiotic also produced by other aerobic pseudomonads (Box et al., 1988; Gwynn et al., 1988).

Tropolone, which is known to have antibacterial and antifungal activities (Lindberg, 1981), is produced by B. plantarii cultures (Azegami et al., 1987).

Plant pathogenicity

Many species of the genus Burkholderia are pathogenic for animals or plants. Phytopathogenic pseudomonads are located in three of the five RNA similarity groups. One of them is rRNA group II, in which Burkholderia and Ralstonia are allocated. The various symptoms produced in plants by the phytopathogenic pseudomonads are tumorous outgrowth, rot, blight or chlorosis, and necrosis, which are caused by alteration of the normal metabolism of plant cells by pathogenicity factors excreted by the bacteria. These factors include enzymes capable of degrading components of plant tissues, toxins, and plant hormones.

The phytopathogenic species of the genus Burkholderia mainly produce rots, due to active pectinolytic enzymes and cellulases (Gehring, 1962; Hildebrand, personal communication). Further details on symptoms and on the list of hosts attacked by each species will be given in the section of species descriptions at the end of this chapter.

Aside from acting as agents of diseases, members of the genus also participate in producing beneficial effects by antagonizing other phytopathogenic organisms, mainly fungi. This effect has been reported in the literature for B. cepacia in a number of instances (Kawamoto and Lorbeer, 1976; Fantino and Bazzi, 1982; Janisiewicz and Roitman, 1988; Homma et al., 1989; Jayaswal et al., 1990; Parke et al., 1991).

Pathogenicity for humans and animals

The most serious animal and human pathogenic species of the genus Burkholderia are B. mallei and B. pseudomallei, the agents of glanders or farcy of equids, and of melioidosis in humans, respectively. Detailed descriptions of these organisms and the diseases that they cause are available (Redfearn et al., 1966; Redfearn and Palleroni, 1975). As a free-living species present in the warm regions of the planet, B. pseudomallei has not spread far from the equator, although in China it has reached a northern latitude of at least 25.5 degrees (Yang et al., 1995b).

A useful updated treatment of the bacteriology of glanders and melioidosis is available (Pitt, 1998). Many years ago, melioidosis was recognized as a glanders-like disease (Whitmore, 1913), and subsequently the organism was assigned to several different genera. In fact, this has also been true of B. mallei, which has spent much of its scientific career in search of a proper generic allocation.

Two different biotypes have been recognized among various strains of B. pseudomallei isolated from patients and from soil in Thailand. However, all of the strains were recognized by using a specific polyclonal antibody. One of the biotypes may have low virulence or it may represent a different species altogether, based on the distribution of these phenotypes and the respective incidence of melioidosis in different areas (Wuthiekanun et al., 1996).

Strains of B. pseudomallei isolated in Australia were examined for their genomic relationships using random amplification of polymorphic DNA and multilocus enzyme electrophoresis. The strains could be divided into two groups that correlated with the clinical presentation and not with the geographic origin; in other words, there was a correlation between the clinical manifestation of the disease and the molecular characteristics of the pathogen (Norton et al., 1998).

Some of the human pathogens are “opportunistic pathogens” that create major medical problems in patients with reduced levels of natural resistance. This situation emerges because of an increasing use of instrumentation or drugs (including antibiotics) that reduce or bypass the level of natural resistance and/or the specific immune mechanisms (Spaulding, 1974).

In reference to B. cepacia, it seems appropriate here to highlight the main points of the abstract of an excellent article published more than a decade ago (Goldmann and Klinger, 1986). From its original habitat as a plant pathogen, B. cepacia has invaded the hospital environment as an important pathogen of compromised human hosts. Many nosocomial infections have their source in contaminated medicaments and even disinfectants and antiseptics. Various conditions in hospital patients can be complicated by B. cepacia infections, but the properties that define the virulence of strains of this species are still poorly defined. The last addition to the long list of pathological conditions that are further deteriorated by infections of this pathogen is cystic fibrosis. As in other conditions, some patients may be simply colonized, while other patients' initial conditions can be very seriously complicated, to which the remarkable resistance of B. cepacia to a wide range of antibiotics contributes very effectively. This bleak panorama has become even worse in the intervening years, particularly with respect to pulmonary exacerbations in cystic fibrosis patients, in many cases ending in rapid and fatal deterioration of lung function.

Characteristics that occur more frequently among strains isolated from infections in cystic fibrosis patients include catalase, ornithine decarboxylase, valine amino-peptidase, lipase, alginase, trypsin, ability to reduce nitrate, hydrolysis of xanthine and urea, complete hemolysis of bovine red blood cells, cold-sensitive hemolysis of human red blood cells, and greening of horse and rabbit red blood cells. Although some of these properties are associated with pathogenicity in other bacteria, their relationships to cystic fibrosis-associated pulmonary disease are far from clear (Gessner and Mortensen, 1990). Acid phosphatase in Burkholderia species is a factor that may be related to pathogenicity. It is present in B. cepacia and B. pseudomallei, and in fact the activity in the latter species is remarkably high (Dejsirilert Butraporn et al., 1989).

In addition to these factors, as mentioned before in the section on antigenic structure, LPS preparations from clinical and environmental isolates of B. cepacia and from the closely related species B. gladioli exhibit a higher endotoxic activity and more pronounced cytokine response in vitro when compared to preparations of P. aeruginosa LPS. The latter species is also involved in infections in cystic fibrosis patients (Shaw et al., 1995).

B. gladioli, a species originally described as phytopathogenic, has been implicated in infections complicating cases of cystic fibrosis (Mortensen et al., 1988). In one study, the organism was isolated from respiratory tract specimens obtained from 11 cystic fibrosis patients and was identified by its biochemical properties, DNA hybridization, and fatty acid analysis. The authors recommend the inclusion of some of these criteria for the differentiation between B. gladioli and B. cepacia, two closely related species, and point out that most B. gladioli strains have C_{10:0 3OH} fatty acid, which is absent from B. cepacia lipids (Christenson et al., 1989). Also based on the results of fatty acid analysis, a pseudomonad that was isolated from pleural fluid and pulmonary decortication tissue with granulomatous disease more closely resembled B. gladioli than B. cepacia (Trotter et al., 1990). Further information on the activity of B. gladioli as a human pathogen has been reported by Ross et al. (1995). A strain of this species was involved in empyema and bloodstream infection occurring after lung transplantation in a cystic fibrosis case (Khan et al., 1996c).

The pili of B. cepacia mediate adherence to mucous glycoproteins (Kuehn et al., 1992; Sajjan and Forstner, 1993) and epithelial cells in cystic fibrosis patients. Structural variant classes of pilus fibers have been identified in B. cepacia strains (Goldstein et al., 1995). One or more of five morphologically different types can be coexpressed. It has been noticed that, when present in the infective population, Pseudomonas aeruginosa cells enhance the adhesion of B. cepacia to epithelial cells (Saiman et al., 1990).

The major pilin subunit of B. cepacia corresponds to peritrichous fimbriae, which, based on their appearance as giant intertwined fibers, have been called “cable” (Cbl) pili. The cblA gene (the first pilin subunit gene to be identified in B. cepacia) has been detected in a DNA library (Sajjan et al., 1995).

One very infectious cystic fibrosis strain isolated as the agent of epidemics in England and Canada was found to have the cable pilin subunit gene. A conserved DNA marker in a 1.4-Kb fragment was present in epidemic strains, absent from the nonepidemic ones, and rare among the environmental strains. The presumed ORF was designated “epidemic marker regulator” (esmR) (Mahenthiralingam et al., 1997). Strains were recovered in cystic fibrosis centers in France. There was cross-colonization in 7 of 13 centers. The most chronically colonized patients harbored a single B. cepacia strain, which suggests a geographically clustered distribution of B. cepacia, with the exception of one genotype. This genotype was detected in four regions and proved to be different from the British-Canadian highly transmissible strain and was able to spread among cystic fibrosis units (Segonds et al., 1997).

Biochemical and genomic properties have been used for the typing of B. cepacia strains of nosocomial origin. In a first step, six enzymes and pigment production subdivided a collection of strains of this species into 12 groups, and the strains from one-third of the collection were further characterized by DNA fingerprinting, ribotyping, and plasmid analysis. By testing the typing scheme on strains isolated years later, the results showed the usefulness and consistency of the genomic typing, and the marked diversity of phenotypes among the strains of the species (Ouchi et al., 1995).

The amplified products of the internal transcribed spacers (ITS) separating the 16S and 23S rRNA genes in the DNA have been effective as tools for identification of reference strains of Pseudomonas aeruginosa and B. pseudomallei, but the primer pairs tested for B. cepacia have not provided much help in strain identification (Tyler et al., 1995). In contrast, PCR-ribotyping targeted on the 16S-23S intergenic spacer to determine the length heterogeneity of this region is a rapid and accurate method for B. cepacia strain typing (Dasen et al., 1994).

The internal diversity of B. cepacia strains is also manifested in differences in whole-cell protein profiles (Li and Hayward, 1994) and in the fatty acid composition of populations from various cystic fibrosis centers (Mukwaya and Welch, 1989).

A novel marker has been found to be associated with epidemic B. cepacia strains causing infections in cystic fibrosis patients. A highly infectious strain had both the cable pilin subunit gene (cblA) and a unique combination of insertion sequences. Although no specific marker was identified in common with other epidemic strains, a conserved DNA fragment among epidemic strains was detected. This fragment (called the “B. cepacia epidemic strain marker”) was absent from other strains infecting individual cystic fibrosis patients and rarely found in environmental strains (Mahenthiralingam et al., 1997).

In a related study on 97 clinical and 2 environmental strains of B. cepacia, a search for possible correlations was made with respect to parameters such as certain insertion sequence (IS) elements, the cblA gene for a pilin subunit, the electrophoretic type (ET), and the ribotype (RT) (Tyler et al., 1996). No linkage was found between the presence of each of five different IS elements and ET or RT. All strains of a given ET also possessed cblA. One IS element different from the five initially identified was present in 72% of all isolates, and in half of them the new IS element was inserted in one of the original five. This hybrid IS element only was found in epidemic strains from Ontario, Canada, and the UK.

Methods were developed to detect the presence of B. cepacia, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia in sputum, using primers based on 16S rRNA sequences. The results are in agreement with those of direct isolation of cultures from the samples (Campbell et al., 1995; Karpati and Jonasson, 1996).

Bongkreik acid (also named flavotoxin A), a product of fermentation by B. cocovenenans, is the cause of serious food poisoning outbreaks in China and other Far East countries (Hu et al., 1989). An isomer, isobongkreik acid, was later identified, and, like bongkreik acid, it acts as an uncompetitive inhibitor of ADP transport in mitochondria, although it is less active than bongkreik acid (Lauquin et al., 1976).

Ecology, habitats, and niches

The habitats from which Burkholderia species have been isolated are quite diverse: soils and rhizospheres (B. cepacia, B. vietnamiensis, B. pseudomallei, B. pyrrocinia, B. phenazinium, B. multivorans, B. thailandensis, B. glathei), water (B. norimbergensis), plants (B. cepacia, B. gladioli, B. caryophylli, B. glumae, B. plantarii, B. andropogonis, B. vandii), foods (B. cocovenenans), animals (B. mallei, B. pseudomallei), and clinical specimens (B. cepacia, B. vietnamiensis, B. gladioli, B. multivorans). The occurrence of B. pseudomallei mainly in soils of tropical regions has been discussed elsewhere (Redfearn et al., 1966).

As mentioned in the section on metabolic properties, some species of the genus are metabolically very versatile, and there is little doubt that those normally found in soils and water must contribute substantially to the mineralization of carbon compounds in nature.

B. cepacia is commonly found in natural materials, particularly soil, and methods have been developed for its detection. One such method of detection in the environment was based on amplification of sequences in genomic DNA using primers specific for repetitive extragenic palindromic segments, followed by cloning of the amplified fragments. Probes were constructed based on the strain-specific sequences (Matheson et al., 1997). A striking example of the power of molecular approaches was the report of the detection of a single B. cepacia cell in a soil sample containing a population of 10¹⁰ procaryotic cells (Steffan and Atlas, 1988). Estimates of the relative abundance of B. cepacia in stream bacterioplankton can be performed after collecting the cells by various procedures, of which the use of filters made of inorganic materials was found to give the highest recoveries (Lemke et al., 1997).

Strains of B. cepacia can be isolated from rhizosphere environments (Tabacchioni et al., 1995). This is also the habitat from which B. graminis was originally obtained (Viallard et al., 1998). Rhizosphere strains of B. cepacia differed in phenotypic characteristics from those isolated from clinical materials. Among the differences were nitrogen fixation, indole-acetic acid production, a wide temperature range, antibiosis vs. phytopathogenic fungi, and growth promotion of Cucumis sativus—all positive for rhizosphere strains. These properties were absent from clinical strains, which instead possessed characteristics such as adhesion to human cells, protease production, and synthesis of siderophores different from those found in the nonclinical strains (Bevivino et al., 1994). The character of N₂ fixation is not clear-cut, since DNA preparations from clinical strains hybridized with the nifA gene probe from Klebsiella pneumoniae, and the DNA of one of the rhizosphere strains hybridized with the nifHDK from Azospirillum brasilense (Tabacchioni et al., 1995).

As far as animal pathogenicity is concerned, observations made in other laboratories seem to support somewhat different conclusions. Thus, strains isolated from plant or clinical materials did not differ in their lethality to mice, i.e., they have similar LD₅₀ values (González and Vidaver, 1979). Indeed, strains of species known to be causal agents of human infections may be isolated from rhizosphere environments (Tabacchioni et al., 1995).

Although B. cepacia helps in the degradation of toxic compounds and has an inhibitory action on soil-borne plant pathogens, it can also behave as a serious opportunistic human pathogen. The key question of interest for its use in biotechnological projects is whether the two types of populations can interact to the point of transmission of the characteristics required for the pathogenic condition. Yohalem and Lorbeer (1994) state that “although there are (B. cepacia) strains with significant potential for the remediation of environmental toxins … and others with potential as biological controls of plant disease … environmental release of any strain may be prohibited because some strains of the nomenspecies have been implicated in nosocomial infections.” The same considerations apply to other versatile species of the genus that manifest pathogenic propensities.

Similarities between Burkholderia and other Gram-negative bacteria

A sharp differentiation of members of Burkholderia and Pseudomonas is difficult because of many similarities between these organisms. In the course of biochemical studies carried out at Berkeley before the rRNA–DNA hybridization experiments showed that “P. multivorans” (B. cepacia) was not closely related to the fluorescent group, it was noticed that key steps of the metabolism of aromatic compounds were remarkably similar between the two groups. In the section on the metabolism of aromatic compounds, some comments have been made on the similarity of components of some of the enzymes involved in the degradation by distantly related organisms. In fact, many details of the metabolic constitution of the fluorescent pseudomonads are far closer to those found in some species of RNA similarity group II—in particular, B. cepacia—than to those characteristic of RNA group V (Stenotrophomonas and Xanthomonas), which is closer to RNA group I. Such is the case of the enzymes codified by genes bphB and bphC in P. putida and B. cepacia, and the same is true for the gamma-carboxymuconolactone decarboxylases of these species. Fluorescent organisms and B. cepacia are able to synthesize salicylic acid, which acts both as a siderophore and also as the precursor of another siderophore found in both groups of organisms, pyochelin. Members of the fluorescent group and B. pyrrocinia, a species related to B. cepacia, produce the antifungal antibiotic pyrrolnitrin, and similarities also have been observed in the pilin antigenic structures of P. aeruginosa and B. cepacia (Saiman et al., 1989).

Similarities have also been observed between B. cepacia and bacteria of groups other than the aerobic pseudomonads. With respect to the production of chaperonins (so named for their relationship with eucaryotic chaperones), an evolutionary homolog of the protein involved in plants in the assembly of ribulose-bisphosphate carboxylase-oxygenase (the key enzyme in CO₂ fixation) was identified in E. coli. This protein, GroEL, is one of the chaperonins, a group widely present in procaryotes and organelles of procaryotic origin (chloroplasts and mitochondria) (Hemmingsen et al., 1988). Aside from E. coli and pseudomonads, chaperonins have been identified in species of Legionella, Bacillus, Borrelia, Treponema, Mycobacterium, and Coxiella (Kaijser, 1975; Houston et al., 1990). In work performed on the cloning and nucleotide sequencing of the groE operons of P. aeruginosa and B. cepacia, a high degree of similarity was found, which extended to the E. coli groEL. The level of similarity with the human protein was lower, but still considerable. Comparable results were obtained in the study of a second chaperonin, GroES (Jensen et al., 1995).

The above-cited findings on chaperonins indicate that the similarities between groups I and II (Pseudomonas and Burkholderia) in some cases go beyond the dispensable catabolic systems or the biosynthesis of secondary products.

Differentiation of Burkholderia from related genera

The examples of similarities mentioned in the previous section show that a sharp differentiation of members of Burkholderia and Pseudomonas is difficult. Studies on DNA–DNA hybridization (Ballard et al., 1970) and on rRNA–DNA hybridization (Palleroni et al., 1973) demonstrated that species of the genus were members of RNA similarity group II. They were eventually allocated to a newly proposed genus under the name Burkholderia (Yabuuchi et al., 1992), but two of the species (B. pickettii and B. solanacearum) were later transferred to the new genus Ralstonia, and the criteria for the differentiation of this genus from Burkholderia were defined (Yabuuchi et al., 1995). Unfortunately, as in the case of the proposal of the genus Burkholderia, the published differential characteristics were limited to those of a single strain of each species, which makes it impossible to estimate the intraspecies diversity.

In a polyphasic study of Burkholderia species, Gillis et al. (1995) redefined this genus. Among the characteristics of differential value, these authors mention the presence of C_{16:0 3OH} in the cellular fatty acid composition. Most of the properties given in the definition of the genus Burkholderia apply equally well to other genera of aerobic pseudomonads, and, as in the case of the genus Pseudomonas, the fatty acid composition and the 16S rRNA characteristics remain among the few useful differential criteria.