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Journal of Plant Pathology (2008), 90 (3), 419-430 Edizioni ETS Pisa, 2008 419

OFFERED REVIEW POLYMYXA: , HYDROLYTIC ENZYMES AND HAZARD ASSESSMENT

W. Raza, W. Yang and Q-R. Shen

1 College of Resource and Environmental Sciences, Nanjing Agriculture University, Nanjing, 210095, Jiangsu Province, P.R. China

SUMMARY pressing several plant diseases and promoting plant growth (Benedict and Langlykke, 1947; Ryu and Park, Certain Paenibacillus polymyxa strains that associate 1997). These strains have been isolated from the rhizos- with many plant species have been used effectively in phere of a variety of crops like wheat (Triticum aes- the control of plant pathogenic fungi and . In tivum), barley (Hordeum gramineae) (Lindberg and this article we review the possible mechanism of action Granhall, 1984), white clover (Trifolium repens), peren- by which P. polymyxa promotes plant growth and sup- nial ryegrass (Lolium perenne), crested wheatgrass presses some plant diseases. Furthermore we present an (Agropyron cristatum) (Holl et al., 1988), lodgepole pine updated summary of antibiotics, autolysis, hydrolytic (Pinus contorta latifolia) (Holl and Chanway, 1992), and autolytic enzymes and levanase produced by this Douglas fir (Pseudotsuga menziesii) (Shishido et al., bacterium. Some hazards and mild pathogenic effects 1996), green bean (Phaseolus vulgaris) (Petersen et al., are also reported, but these appear to be strain-specific 1996) and garlic (Allium sativum ) (Kajimura and Kane- and negligible. The association between plants and P. da, 1996). P. polymyxa has been successfully used to con- polymyxa seems to be specific and to involve co-adapta- trol Botrytis cinerea, the causal agent of grey mould, in tion processes. There is every reason to believe that strawberries (Helbig, 2001), Fusarium oxysporum and gaining an improved understanding of these processes Pythium spp. causal agents of seedling blight, wilt and will enhance and facilitate efforts to wean off farmers root rot of cucumber and water melon (Yang et al., 2004; dependence on a wide range of agricultural chemicals. Dijksterhuis et al., 1999) sesame damping off (Ryu et al., 2006), and to control diseases of arabidopsis caused by Key words: rhizosphere, plant association, plant Phytophthora palmivora and Pythium aphanidermatum growth promotion, enzymes. (Timmusk and Wagner, 1999). P. polymyxa is known to produce two types of peptide antibiotics, one type is on- ly active against bacteria and the other is active against INTRODUCTION fungi, gram positive bacteria and actinomycetes (Beatty and Jensen, 2002). This species also synthesizes plant Among the myriads of bacteria thriving in the plant hormones auxin (Lebuhn et al., 1997) and cytokinin rhizosphere, some spore-forming plant-growth-promot- (Timmusk et al., 1999), solubilizes soil phosphorus ing rhizobacteria (PGPR), in particular gram-positive (Singh and Singh, 1993), enhances soil porosity (Gouzou and streptomycetes, attracted special attention et al., 1993) and has been used for flocculation and flota- due to their advantages over non-spore formers in prod- tion of various minerals (Deo and Natarajan, 1998). P. uct formulation and stable maintenance in soil (Emmert polymyxa spores cause sporangium deformation and and Handelsman, 1999). Among these, the genus Paeni- have thick walls with a star-shaped section. These can re- (species of a genus previously included in the main in a dormant state for long periods, being resistant genus Bacillus, Ash et al., 1993; Truper, 2005), compris- to heat, drying, radiation and toxic chemicals (Comas- es more than eighty-nine species up to the time of writ- Riu and Vives-Rego, 2002). This antagonistic potential is ing this article (February 2008). Paenibacillus species the base for effective applications of P. polymyxa strains are facultatively anaerobic, endospore-forming, low as an alternative to the chemical control against a wide G+C Gram-positive bacilli (http://www.bacterio.cict.fr/ set of fungal and bacterial plant pathogens. p/paenibacillus). Strains of Paenibacillus polymyxa (the The aim of this review is to evaluate and discuss the type species of genus), were found to be capable of sup- possible updated mechanisms that enable P. polymyxa strains to control a variety of pathogens that invade plants. This review also covers the effective use of these Corresponding author: Q-R. Shen Fax: +86-25-84431492 strains as biofertilizer and eventual hazards, if any, E-mail: [email protected] caused by P. polymyxa. 002_OfferedReview_419 13-11-2008 14:35 Pagina 420

420 Properties and hazard assessment of P. polymyxa Journal of Plant Pathology (2008), 90 (3), 419-430

Fig. 1. A. Scanning electron micrograph of cells of Paenibacillus polymyxa isolate B2 (Timmusk et al., 2003). Whole view (B) and cross- section C) of P. polymyxa spores (Dondero and Holbert, 1957). Bar = 2 µm.

ANTIBIOTIC COMPOUNDS isms. The first member of the family, polymyxin B, was discovered in 1947 (Ainsworth et al., The main type of peptide antibiotics produced by 1947; Stansly et al., 1947). Since then, at least 15 differ- some but not all strains of P. polymyxa is the polymyxin- ent have been discovered in diverse bacteri- -circulin family (Shoji et al., 1977a, 1977b, al species, but the genetic control of their biosynthesis 1977c; Umezawa et al., 1978). Other strains produce has been described only in part. The general structure different peptides, including polypeptins (Doi and Mc- of polymyxins includes a cyclic heptapeptide moiety at- Gloughlin, 1992), jolipeptin (Ito and Koyama, 1972a, tached to a tripeptide side chain with fatty acyl residues 1972b), gatavalin (Nakajima et al., 1972), gavaserin on the N-terminal amino group (Fig. 2). Polymyxins can (Pichard et al., 1995), saltavidin (Pichard et al., 1995), be distinguished from each other by differences in fusaricidins (Kajimura and Kaneda, 1996, 1997) and amino acid or fatty acid composition (Orwa et al., polyxin (Piuri et al., 1998). In addition, P. polymyxa 2002). Polymyxin B and polymyxin E (colistin) have NRRL-B-30509 secreted bacteriocin which reduced been extensively characterized and are being used as both levels and frequency of chick colonization by sulfate, sulfomethylated or colistimethate forms of med- Campylobacter jejuni (Stern et al., 2005; Svetoch et al., 2005). P. polymyxa also produced some antimicrobial and antifungal compounds whose nature remained until now undefined (Rosado and Seldin, 1993; Oedjijono et al., 1993; Liang et al., 1996; Dijksterhuis et al., 1999). Pathogen (mainly fungi) suppression takes place by means of an interaction process, in which the presence of living bacteria is a prerequisite for continuous sup- pression. It was found that anti-fungal compounds pro- duced by the bacteria were counteracted by magnesium ions (Dijksterhuis et al., 1999).

Polymyxin-Colistin-Circulin Family Polymyxins have a bactericidal effect on Gram-nega- tive bacilli, especially Pseudomonas and coliform organ- Fig. 2. General structure of polymyxins. 002_OfferedReview_419 13-11-2008 14:35 Pagina 421

Journal of Plant Pathology (2008), 90 (3), 419-430 Raza et al. 421

icines especially for infections caused by Gram-negative 219 protoplasts is immediately initiated by the addition bacteria. The major types of polymyxin B are polymyxin of jolipeptin, but not by colistin, even at higher concen- B1- B6 (Thomas et al., 1980; Orwa et al., 2001). Anoth- trations, which, however, lyses spheroplasts of Gram- er minor type described so far, is isoleucine-polymyxin negative bacteria. Colistin is inactivated by Nagarse and B1 (Elverdam et al., 1981). Polymyxin E is effective colistinase, which have no effect on jolipeptin (Ito and against Gram-negative bacilli, except Proteus. The ma- Koyama, 1972a, 1972b). jor types of polymyxin E are polymyxin E1 and E2. Mi- Polyxin is a proteinaceous factor with a bacteriolytic nor types include polymyxins E3, E4 and E7 (Thomas effect on Gram-positive bacteria, and bacteriostatic ef- et al., 1980; Orwa et al., 2001), norvalinepolymyxin E1, fect on actively growing Gram-negative cells (Piuri et valine-polymyxin E2 (Elverdam et al., 1981), valine- al., 1998). These differences in reaction of polyxin be- polymyxin E1, isoleucine-polymyxin E1, isoleucine- tween the two classes of organisms might depend on polymyxin E2 (Ikai et al., 1998) and isoleucine- differences in cell wall structure. polymyxin E8 (Orwa et al., 2001). Polymyxins alter cytoplasmic membrane permeability Polymyxin A, B, C and D have differences of little sig- by binding to a negatively charged site in the nificance in the molecular weights but at high surface lipopolysaccharide layer that has an electrostatic attrac- concentration. The characteristics of their unimolecular tion for the positively charged amino groups in the films when spread at the air-water interface on concen- cyclic peptide portion. The polymyxin fatty acid moiety trated salt solution, can classify polymyxins according to dissolves in the hydrophobic region of the membranes the compressibility, collapse pressures and electrical and disrupts their integrity. This causes leakage of cellu- properties. In these respects, polymyxins A and D have lar molecules, inhibition of cellular respiration and identical force-area curve values whilst films of polymyx- binding and inactivation of endotoxin (Teuber and Bad- in B only appear to be slightly more closely packed than er, 1976; Wiese et al., 1998). Polymyxins seem to be those of polymyxin E. Moreover, under conditions of nonspecific for cell membranes of any type and are equal surface pressure the molecules of polymyxin B and highly toxic. Although the structure of most polymyxins E have closer surface packing than those of polymyxins produced by P. polymyxa has been determined, some of A and D. It is interesting to note that polymyxins A and them require further studies. D also possess similar pharmacological action since both produce severe toxic activity (proteinuria) in animals, Fusaricidin-type compounds whereas polymyxins B and E elicit only slight toxic ef- fects (Few and Schulman, 1953). Fusaricidins compose a group of cyclic depsipeptides Polymyxin B has two hydrophobic regions, the fatty that have Mr 883, 897, 948, and 961 Da, with an unusu- acid moiety at the N terminus (6-methylheptanoic/oc- al 15-guanidino-3-hydroxypentadecanoic acid moiety tanoic-Dab) and the D-Phe5-Leu6 segment in the pep- bound to a free amino group (Kajimura and Kaneda, tide ring. The removal of the fatty tail by proteolytic 1996, 1997) (Fig. 3). Fusaricidins are produced by P. cleavage gives rise to a product denoted polymyxin B polymyxa PKB1 at the beginning of sporulation (Beatty nonapeptide, which has no direct antimicrobial proper- and Jensen, 2002) and gatavalin is produced by B. ties, reduces membrane permeabilization, neurotoxicity polymyxa ssp. colistinus koyama (ATCC 21830) (Nakaji- and nephrotoxicity (Chihara et al., 1973; Duwe et al., ma et al., 1972). Both products belong to the same or a 1986; Ouderkirk et al., 2003) but retains the anti-endo- similar family of peptide antibiotics with very similar toxin activity and increases the permeability of the outer HPLC profile. However, no further structural informa- membrane of Gram-negative bacteria towards hy- tion on the gatavalin family of peptides has since ap- drophobic antibiotics (Danner et al., 1986). Colistin can peared. Recently, Raza et al. (unpublished information) also be degraded to a nonapeptide that lacks the fatty have isolated another strain of P. polymyxa SQR-21 that acid moiety and is approximately 15-fold less toxic than produced fusaricidins A-D. the parent compound (Warren et al., 1985). Kurusu et al. (1987) reported the production by P. Two other polymyxins, S1 and T1, isolated from P. polymyxa of a series of peptides designated as LIF03, polymyxa Rs-6 and E-12, respectively, are strongly basic LI-F04, LI-F05, LI-F07, and LI-F08. Although no com- substances primarily active against Gram-negative bac- plete structural analyses were conducted, mass spectro- teria, though polymyxin T1 exhibits a higher activity scopic analyses indicated that LI-F04, one of the major against Gram-positive bacteria than other polymyxin products, was actually a mixture of two peptides with antibiotics (Shoji et al., 1977a, 1977c). P. polymyxa var. Mr 883 and 897 Da and LI-F03 was a mixture of two colistinus synthesizes jolipeptin, a polypeptide peptides with Mr 948 and 961 Da. Upon a closer study which inhibits growth of Gram-positive and Gram-neg- of HPLC profiles and molecular weights of these relat- ative bacteria and contains glycine, alanine, serine, va- ed antibiotics, the antifungal material from PKB1, line, glutamic acid and 2,4-diaminobutyric acid but no gatavalin and LI-F04 appeared to be mixtures of fusari- fatty acids, as colistin does. The lysis of B. subtilis PCI cidins A and B; LI-F03 was a mixture of fusaricidins C 002_OfferedReview_419 13-11-2008 14:35 Pagina 422

422 Properties and hazard assessment of P. polymyxa Journal of Plant Pathology (2008), 90 (3), 419-430

and D; LI-F05 was a mixture of fusaricidin A and an uncharacterized fusaricidin-type peptide; and LI-F07 and LI-F08 could also be mixtures of other as yet un- characterized fusaricidin-type peptide antibiotics. Two further antibacterial substances were isolated and purified from culture broth of P. polymyxa. The first compound, named gavaserin (911 Da), contains glutamic acid, alanine, valine, serine, 2,4-diaminobutyric acid and octanoic acid. The second compound, named saltavalin (903 Da), contains serine, alanine, leucine, threonine, valine and 2,4-diaminobutyric acid, but no fatty acid (Pichard et al., 1995). The fusaricidin group of antibiotics seems to be less Fig. 3. Structure of fusaricidins A, B, C, and D. Adapted from diverse as compared with the Polymyxin-Colistin-Cir- Umezawa et al. (1978) and Kajimura and Kaneda (1996, culin group. However, both groups still lack complete 1997). structural and molecular characterization. and their effectiveness as biological control agents is still Undefined compounds matter of conjecture. What reported above reflects the fact that antibiotic production is a frequently encoun- P. polymyxa strains also produce many undefined an- tered, but non-uniform characteristic of P. polymyxa timicrobial compounds. One such antibiotic having a strains. It is also noticeable that the Polymyxin-Colistin- Mr of less than 3,500 Da is active against the majority of Circulin family of antibiotics is effective against Gram- Gram-positive bacteria but lacks activity against negative bacteria, while Fusaricidin-type antibiotics are , a species usually killed by effective against Gram-positive bacteria. There are, how- polymyxins (Rosado and Seldin, 1993). An unknown ever, also some compounds produced by P. polymyxa antimicrobial peptide (2,983 Da) was described by which are effective against both Gram-positive and -neg- Zengguo et al. (2007) as a novel antibiotic with a high ative bacteria, such as polyxin. Newly described antibi- degree of post-translational modifications, showing ac- otics, having similar structure, but different antifungal tivity against Gram-positive food-borne pathogenic and and antibacterial activity, require regrouping of antibi- spoilage bacteria. Two other compounds, with Mr 1,184 otics produced by P. polymyxa strains. The production and 1,202 Da, respectively, had a structure very similar of different kinds of antibiotics that are effective against to that of polymyxin B1, with a cyclic heptapeptide moi- different types of pathogens show the need for careful ety attached to a tripeptide side chain and a fatty acyl selection of P. polymyxa strains antagonistic to specific residue, but were more hydrophobic than polymyxin B. plant pathogens, to ensure effective control. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184 Da, containing a 2,3-didehy- Non-ribosomal synthesis of antimicrobial peptides drobutyrine residue with Mr 101 Da (replacing a threo- Molecular and genetic aspects regarding antibiotic nine at position 2 of the polymyxin side chain) was re- synthesis by Paenibacillus spp. are still partially known. ported by Selim et al. (2005). The characterization of The biosynthesis of mattacin by Paenibacillus kobensis these two peptides confirmed their broad spectrum an- M was examined by Martin et al. (2003), using transpo- tagonistic activity against Gram-positive bacteria, in sitional mutagenesis by which 10 production mutants contrast to polymyxin B. The latter modifications could were obtained, revealing a set of genes involved in its be the cause of broader range of antagonistic activity of production. The majority of the mutants exhibited de- these peptides compared to that of polymyxin B. creased levels of production as opposed to an expected complete loss of production. This is not consistent with General comments on antibiotic production other studies using Tn917 for iturin and fengycin by P. polymyxa biosynthesis genes, where complete loss of antibiotic production was observed (Chen et al., 1995; Tsuge et Soil microbial production of secondary metabolites al., 2001). The identified genes of P. polymyxa showed that can promote or constrain plant growth has often low homology to known genes in the NCBI database. P. been found to be finely tuned, controlled by environ- polymyxa strain SCE2 was transformed with plasmid mental conditions, medium components, and influenced pTV32 (Ts), a delivery vector for Tn917-lac. After trans- by root activities. Such information regarding antibiotics position, four mutants were shown to have lost their ca- of P. polymyxa is not yet available. In addition, the corre- pability to inhibit Micrococcus sp. and Staphylococcus au- lation between the production of antibiotics by an isolate reus RN450, but they continued to inhibit the growth of 002_OfferedReview_419 13-11-2008 14:35 Pagina 423

Journal of Plant Pathology (2008), 90 (3), 419-430 Raza et al. 423

Corynebacterium fimi NCTC7547 and Escherichia coli domain is found in the sixth module, corresponding to HB101. It was found that the place of insertion of D-Ala. This sixth adenylation domain was produced at Tn917-lac in the chromosome was the same in mutants a high level in Escherichia coli and was shown to activate 4 and 36 and in mutants 31 and 59 but different be- D-Ala specifically, providing evidence for direct activa- tween these pairs. It was therefore thought possible that tion of a D-amino acid by a prokaryotic peptide syn- more than one antimicrobial substance could be pro- thetase. The fusaricidin gene cluster also includes genes duced by strain SCE2 (Seldin et al., 1999). Recently, a involved in the biosynthesis of the lipid moiety, but no DNA fragment that appears to encode the first two genes for resistance, regulation, or transport functions modules of the putative fusaricidin synthetase (fusA) were encountered (Li and Jensen, 2008). was isolated from P. polymyxa PKB1. To confirm the in- The production of nonapeptide or octapeptide an- volvement of fusA in the production of fusaricidins, a tibiotics, following removal of the fatty acid tail, de- modified PCR-targeting mutagenesis was done to create creases their direct antimicrobial and permeabilization a fusA mutation in PKB1. Targeted disruption of fusA activity. Natural mutations by unknown sources may in- led to the complete loss of antifungal activity against activate some enzymes involved in antibiotic produc- Leptosphaeria maculans suggesting that fusA plays an es- tion, so products could be nonapeptides or octapep- sential role in the nonribosomal synthesis of fusaricidins tides, with or without fatty acid residues. Further stud- (Li et al., 2007). Afterward, the fusaricidin biosynthetic ies are required to understand why mutants have partial gene cluster was cloned and sequenced. It spans 32.4 antibiotic activity. It is clear that P. polymyxa strains pro- kb, including an open reading frame (fusA) encoding a duce many different antimicrobial compounds reflect- six-module nonribosomal peptide synthetase. The sec- ing their genetic diversity and that more (medium de- ond, fourth, and fifth modules of fusaricidin synthetase pendent) compounds may be discovered in the (near) each contain an epimerization domain, consistent with future. The non-ribosomal peptide synthetase (NRPSs) the structure of fusaricidins. However, no epimerization and polyketide synthetases (PKSs) are extremely

Table 1. List of antifungal and antibacterial compounds produced by different strains of Paenibacillus polymyxa.

Name Molecular weight (Da) References Bacteriocin 3864 Stern et al., 2005 Fusaricidin A 883 Kajimura and Kaneda, 1996 Fusaricidin B 897 Kajimura and Kaneda, 1997 Fusaricidin C 947 Kajimura and Kaneda, 1997 Fusaricidin D 961 Kajimura and Kaneda, 1997 Gatavalin >3500 Nakajima et al., 1972 Gavaserin 911 Pichard et al., 1995 Jolipeptin - Ito and Koyama, 1972a, 1972b LI-F03, 961, 947 Kurusu et al., 1987 LI-F04 897, 883 Kurusu et al., 1987 LI-F05 911, 897 Kurusu et al., 1987 LI-F07 945, 931 Kurusu et al., 1987 LI-F08 925, 911 Kurusu et al., 1987 Polymyxin A - Catch et al., 1948, 1949

Polymyxin B1 1202.48 Ainsworth et al., 1947

Polymyxin B2 1184.7 Benedict and Langlykke, 1947 Polymyxin C - John, 1948, 1949 Polymyxin D 1144.37 Bell et al., 1949

Polymyxin E1 (Colistin A) 1169.46 Elverdam et al., 1981

Polymyxin E2 (Colistin B) 1155.43 Elverdam et al., 1981 Polymyxin M 1157.41 Silaev et al., 1975

Polymyxin S1 1178.38 Shoji et al., 1977b

Polymyxin T1 1215.58 Shoji et al., 1977c Polypeptin A 1115.41 Doi and McGloughlin, 1992 Polyxin 10,000 Piuri et al., 1998 Saltavalin 903 Pichard et al., 1995 002_OfferedReview_419 13-11-2008 14:35 Pagina 424

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amenable to genetic manipulations, providing powerful dates for the biological control of pathogenic fungi. tools for future development and production of novel The endophytic P. polymyxa strain GS01 was isolated peptides, polyketides and hybrid compounds with new from the interior of the roots of Korean cultivars of gin- properties. Increased knowledge about regulation of the seng (Panax ginseng). The gene cel44C-man26A cloned antibiotic production and formation of other products from this strain contained a glycosyl hydrolase family 44 can be used as guidelines in order to enhance antibiotic (GH44) catalytic domain, a fibronectin domain type 3, a production. Further research is required to locate glycosyl hydrolase family 26 (GH26) catalytic domain, biosynthesis assembly of secondary metabolites and an- and a cellulose-binding module type 3. The multifunc- tibiotics and assessing their role at the molecular and tional enzyme domain GH44 possessed cellulase, xy- genetic level for the persistence or survival of P. lanase, and lichenase activities, while the domain GH26 polymyxa in the diverse environment of the rhizosphere had mannanase activity (Cho et al., 2006). and its effect on other microbial communities. A Paenibacillus sp. strain denoted BL11, was found to secrete pectate lyase, a pectin degrading enzyme, whose gene, cloned from BL11, was 963 nucleotides in HYDROLYTIC ENZYMES size and encoded a protein of 38 kDa. Because of its good pectate lyase activity, the bacterial stain in ques- P. polymyxa strains are capable to produce several hy- tions has the potential for industrial application drolytic enzymes that play an important role in the bio- (http://etds.lib.ntu.edu.tw/etdservice/view_metadata?et control of plant pathogens (Dijksterhuis et al., 1999; dun=U0001-2507200616122300&query_field1=key- Helbig, 2001; Yang et al., 2004). Sakurai et al. (1989) word&&query_word1=REE&). found that P. polymyxa 72 produced an amylase of 48 The thermal stability of b-glucosidase encoded by the kDA that comprises 1,161 amino acids. Its gene ap- bglA gene from P. polymyxa was improved. The wild peared to be divided into two parts by a direct-repeat type enzyme has a half-life of 15 min at 35°C and no de- sequence located almost centrally in the gene. The 5’ re- tectable activity at 55°C. Random mutagenesis resulted gion upstream the direct-repeat sequence was shown to in enhanced tolerance to higher temperatures of the be responsible for the synthesis of beta-amylase. The 3’ mutants, viz. a half life of 12 min at 65°C (Zhao and region downstream the direct-repeat sequence con- Arnold, 2000). tained four sequences homologous with those of other A sacB gene encoding levansucrase, cloned and se- alpha-amylases, such as Taka-amylase A. quenced from P. polymyxa CF43, increased the mass of P. polymyxa can produce heat stable proteases (30 root-adhering soil. This P. polymyxa gene was homolo- kDa), constitutive or partially inducible in nature and gous to a similar gene of that synthesizes strongly influenced by variation in carbon/nitrogen ratio high molecular weight levan (Bezzate et al., 2002). or by the presence of some easily metabolizable sugars The P. polymyxa CF43 lelA gene, expressing both su- (Alvarez et al., 2006). A protease of this type, produced crose and fructan hydrolase activities, was isolated from by P. polymyxa B-17, had a temperature optimum at a genomic P. polymyxa library screened in B. subtilis. 50°C and retained significant activity at 70°C, was active This gene expressed sucrose hydrolase activity. The de- at pH ranging from 5.5 to 10.0, and was inhibited by duced amino acid sequence showed 54% identity with metal chelating agents (Matta and Punj, 1998). mature B. subtilis levanase and was similar to other fruc- Three xylanases were purified to homogeneity from tanases and sucrases (Pi-D-fructosyltransferases) (Bez- the extracellular enzymatic complex of P. polymyxa. zate et al., 1994). These enzymes were small proteins of 34, 34, and 22 Lectins LI and LII from P. polymyxa 1460, showed kDa in size and basic pIs were 9.3, >9.3 and 9.0, respec- an increase in their proteolytic activity when incubated tively (Morales et al., 1993). Chitinase and b-1,3-glu- with the carbohydrate moiety of the wheat-root exo- canase were produced when P. polymyxa was grown in component fraction. This increase could be associated the presence of colloidal chitin as the sole carbon source with the presence of lectin-specific carbohydrates in the (Mavingui and Heulin, 1994). root fraction. The lectins of the nitrogen-fixing paeni- Production of hydrolytic enzymes is expected to be bacilli enhanced cellulose degradation in the plant cell, affected by the presence of monosaccharide products in thus increasing the activity of beta-glucosidase in the the growth media. However, in a comparative trial for wheat-root cell wall (Karpunina et al., 2003). The gene enzyme production in PA and PDA media, both cellu- encoding the neopullulanase enzyme from P. polymyxa lase and mannanase activities of two P. polymyxa strains CECT 155 was expressed in B. subtilis and the corre- were unaffected by glucose, indicating medium-inde- sponding transformants produced extracellular neopul- pendent production (Nielsen and Sorensen, 1997). Such lulanase (Yebra et al., 1999). medium-independent expression of enzymes for degra- P. polymyxa appears to produce many hydrolytic en- dation of cellulose-containing cell wall components in- zymes, but further characterization and isolation of dicates these P. polymyxa strains as promising candi- strains or mutants which can produce heat stable or 002_OfferedReview_419 13-11-2008 14:35 Pagina 425

Journal of Plant Pathology (2008), 90 (3), 419-430 Raza et al. 425

more heat-resistant hydrolytic enzymes should be per- non-inoculated rhizosphere soil. In its natural environ- formed to ensure maximum survival of bacteria under ment, P. polymyxa population remained at a constant adverse climate conditions and attack of pathogens. The level (Gouzou et al., 1993). In contrast, inoculation with detailed modes of regulation need also to be thoroughly P. polymyxa mutant strain SB03 had no effect on root- characterized. The physiological impacts of these genes adhering soil mass, compared with the non-inoculated need to be better understood and the genes whose ex- treatment. P. polymyxa SB03 is a mutant whose gene en- pression is altered (either up or down) need to be elabo- coding the enzyme for levan synthesis, sacB was inacti- rated in detail including an understanding of how the vated. Thus, results strongly suggest that levan synthesis proteins encoded by these genes contribute to plant by strain CF43 is the main mechanism involved in the growth and development. improvement of root-adhering soil structure (Bezzate et al., 2000). Reasons for soil aggregation may be several, i.e. culti- AUTOLYSIS var specificity for the establishment of a productive as- sociation between host and bacteria, a too high nutrient A 23 kDa autolysin (cwlV) purified from P. polymyxa availability at the site, or the threshold of inoculum den- var. colistinus exhibited cell wall hydrolytic activity. The sity, apart from other possible physicochemical factors. cwlV consists of a 1,497 bp ORF that encodes a The influence on soil structure should be further inves- polypeptide of 499 amino acid residues. Identification tigated not only with reference to the above issues, but of the specific substrate bond cleaved by the autolysin also in relation to spatial extension of the rhizosphere indicated that the enzyme is an N-acetylmuramoyl-L- soil. Inoculation by P. polymyxa could play an important alanine amidase. Optimal reaction temperature was 40- role in water retention and nutrient transfer in the rhi- 50°C and optimal pH 4.0, which is unique for amidases zosphere by increasing porosity. (Kuroda and Sekigocbi, 1990; Oda et al., 1993). The alignment of the N-terminal 18 amino acid sequence of cwlV with N-terminal sequences of the catalytic domain POSSIBLE HAZARDS CONNECTED WITH of class II amidases suggested that cwlV might belong to THE USE OF P. POLYMYXA IN BIOCONTROL class II amidases. The cwlV enzyme did not significantly hydrolyze cell walls of and B. subtilis P. polymyxa may have adverse effects on plants. For (Kawahara et al., 1997). This seems to support earlier example, when roots of Arabidopsis thaliana were findings that the cell wall structure of P. polymyxa is dif- soaked for 24 h in cultures of P. polymyxa strains B2, B3 ferent from that of Micrococcus luteus and B. subtilis. and B4 in L medium, plants responded with 30% Autolysis activity may be a self-control mechanism of growth reduction and a stunted root system. These ef- the bacterial population in this case, but further re- fects were observed in a gnotobiotic system and in the search is required to find out possible reasons and opti- soil (Timmusk and Wagner, 1999). Marker genes for mum conditions for autolysis. Sequencing of the flank- Ethylene (ET), Jasmonic acid (JA) and Salicylic acid ing region revealed that another putative autolysin gene, (SA) pathways, i. e. HEL [heveine-ET pathway (Potter cwlU, was located upstream cwlV. The cwlU encoded a et al., 1993)], ATVSP [vegetative storage protein acid polypeptide of 524 Da, its deduced sequence was phosphatase-JA pathway; (Berger et al., 1995)], and PR- 34.9% identical to the full-length sequence of cwlV. It 1 [SA pathway (Uknes et al., 1992)], were over-ex- was found that cwlU transcribed as a single cistron, pressed in plants exposed to P. polymyxa at variable in- whereas cwlV transcribed with orfW. The analysis of duction levels (Timmusk et al., 2003). Furthermore, in- unprocessed forms of cwlV and cwlU (VdeltaS and oculation with P. polymyxa strain L6-16R promoted UdeltaS, respectively) and their predicted mature forms growth of lodgepole pine in one location, inhibited it in (Vcat and Ucat, respectively) indicated that VdeltaS and a second site, and had no discernible effect in a third Vcat exhibited low and high cell wall hydrolase activi- site (Chanway and Holl, 1994). ties, respectively, towards P. polymyxa cell walls, but In conclusion, it appears that under particular exper- UdeltaS and Ucat had almost no and low cell wall hy- imental conditions P. polymyxa can behave either as a drolase activities, respectively (Ishikawa et al., 1999). deleterious rhizobacterium (DRB) or, since it induces the formation of around the root tip, as a root- invading bacterium (Timmusk et al., 2005). The popula- SOIL AGGREGATION tion density of rhizosphere-colonizing and root-invading bacteria differs in the gnotobiotic and soil systems due P. polymyxa was found to increase the mass of soil to the different environmental conditions. Nevertheless, adhering to the wheat roots by 57%. Comparison of ag- P. polymyxa has two preferential colonization sites, the gregate size distributions suggested that there was a first located at the root tip in the elongation zone, which more porous structure in the inoculated than in the sometimes results in the loss of the root cap, the other 002_OfferedReview_419 13-11-2008 14:35 Pagina 426

426 Properties and hazard assessment of P. polymyxa Journal of Plant Pathology (2008), 90 (3), 419-430

in the differentiation zone, possibly because these root CONCLUDING REMARKS regions are rich nutrient sources (Jaeger et al., 1999; Timmusk and Wagner, 1999; Timmusk et al., 2003). This review focuses on the possible mechanism of ac- The mechanisms by which DRB sometimes promote tions that enable certain P. polymxa strains to control a plant growth are poorly understood. It is possible that wide range of plant pathogens and to have a restrictive the damage caused by the bacterium to the root tip ulti- effect on the development of certain plant diseases. mately results in activation of defense genes. This is in These strains were found to produce a variety of antibi- line with the up-regulation of the PR-1, ATVSP, and otics (not all of which are well characterized) effective HEL genes that are associated with biotic stresses and against a range of organisms. The correlation between the ERD15 gene, associated with drought responses. A antibiotic production by an isolate and its effectiveness second possibility is that a given P. polymyxa isolate as biological control agent is, unfortunately, still matter could be a potentially aggressive biocontrol agent for use of conjecture. Research activity in this field over the last against pathogens that colonize the root tip. A third pos- decade has been remarkable and the interest continues sibility is that the P. polymyxa formed on the unabated now, especially after recent evidence of the in- roots coincides with the colonization sites of several duction by P. polymyxa strains of localized and systemic pathogens and thereby functions as a protective layer to resistance to a variety of plant pathogens in a range of prevent access by pathogens. The protection layer might plants. In addition, the study of P. polymyxa strains as a also contribute to the plant-enhanced drought tolerance. source of biologically active metabolites is especially sig- Differences in the physiological characteristics of nat- nificant and is likely to focus the attention on this subject ural isolates of the same species have been observed for years to come. The main interest lies in compounds (Lebuhn et al., 1997), which may reflect the selection of that exhibit antibiotic activity because they are more like- subpopulations of P. polymyxa by specific conditions of ly to be implicated in the effectiveness of bacterial strains the rhizosphere environment. Thus, we suggest that dif- used in biological control. P. polymyxa strains produce ferent P. polymyxa isolates may exist, that are indistin- many hydrolytic enzymes, making them valuable antago- guishable based on their 16S rRNA gene sequences, but nists to control plant pathogens and reflect the potential behave differently with respect to gfp expression, plas- of these strains for further industrial application. mid maintenance and growth rate. Soil microbial com- The association between plants and P. polymyxa seems munity study should be conducted to look for the pres- to be specific and to involve co-adaptation processes. A ence of such strains. selection at the subspecies level of P. polymyxa may be To overcome the inefficiency of P. polymyxa as a bio- part of the interdependence of plant roots and the popu- control agent under some environmental conditions, lation structure and a function of the root-associated soil combinations of antagonists with different modes of ac- microflora. Co-inoculation of P. polymyxa with other PG- tions, may allow greater consistency in pathogens con- PRs can increase biocontrol activity due to different trol. A strain that interacts with a host plant as a plant mode of actions and survival abilities under diverse envi- growth promoting rhizobacteria (PGPR) or a DRB is ronmental conditions. Interestingly, P. polymyxa strains strongly dependent on the prevalent environmental con- are active against a wide range of fungi and bacteria but ditions which may determine whether beneficial or dele- not against arbuscular mycorrhizae. terious aspects dominate. This duality could be the re- The negative effects of some P. polymyxa strains, e.g. sult of subtle alterations in gene regulation or horizontal root invasion, appear to be mostly strain-specific and gene transfer. For example, minor introduction of ge- negligible as compared with beneficiary affects e.g. nomic DNA (insertions by recombination or mutations) growth promotion and pathogen control. Interaction of can convert a pathogen to a saprophyte and vice versa a bacterial strain with a host plant as a PGPR or a DRB (Hsia et al., 1997). Further research is required to find is strongly dependent on the prevalent rhizosphere envi- out possible limits of beneficial and deleterious effects ronment conditions. In any case, the role of P. polymyxa of P. polymyxa under particular conditions. in the rhizosphere microbial community requires fur- A single case of P. polymyxa-elicited bacteremia was ther studies, also because there is every reason to be- reported in a patient with cerebral infarct in Japan (Na- lieve that gaining a greater understanding of these su et al., 2003). Although P. polymyxa does not appear processes will facilitate in the long range the efforts to to possess virulence factor genes of its own, acquistion wean off dependence on agricultural chemicals. of genes of this kind through horizontal transfer from other bacteria is theoretically possible. However, to the best of our knowledge, the above is the only report of P. REFERENCES polymyxa as human pathogen, so its significance ap- pears to be negligible. Ainsworth G.C., Brown A.M., Brownlee G., 1947. Aerosporin, an antibiotic produced by Bacillus aerosporus Greer. Nature (London) 160: 263. 002_OfferedReview_419 13-11-2008 14:35 Pagina 427

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Received March 11, 2008 Accepted June 20, 2008