N2-fixing pseudomonads and related soil bacteria

FEMS Microbiology Reviews 13 (1994) 95-118 © 1994 Federation of European Microbiological Societies 0168-6445/94/$26.00 Published by Elsevier

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F E M S R E (10366

N2-fixing pseudomonads and related soil bacteria Y i u - K w o k C h a n * " , W i l f r e d o L. B a r r a q u i o b and R o g e r K n o w l e s c " Plant Research Centre, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario, Canada K1A 0C6, h Microbiology

Laboratory, Institute of Biology, University of the Philippines, Diliman, Quezon Ci~ 1101, Philippines, and c Microbiology Unit, Department of Natural Resource Sciences, Macdonald Campus of McGill University, 21111 Lakeshore Road, Ste. Anne de Bellel:ue, Quebec, Canada HgX 3V9 (Received 8 April 1993; accepted 20 October 1993)

Abstract: Pseudomonas-like organisms form a highly heterogeneous and ubiquitous group of bacteria. Recent identification of an authentic P~eudomonas genus should help to decrease difficulties which arose from taxonomic uncertainties. Further difficulties in recognising N2-fixing Pseudomonas species lie in the confirmation of diazotrophy. Optimised conditions are often needed for the detection of nitrogenase activity since it is controlled by specific environmental factors and physiological requirements. However, genetically constructed N2-fixing strains from authentic Pseudomonas species have demonstrated that at least some members of the genus possess mechanisms to accommodate and express nil (N z fixation) genes from a well-studied diazotroph, Klebsiella pneumoniae. Renowned for their catabolic versatility, pseudomonads can use a wide range of carbon and energy substrates. Hence, potential N2-fixing pseudomonads are conceivably less limited by carbon and energy sources available in the environment compared to other N2-fixing organisms. Pseudomonas species dominate in the rhizosphere of some plants from which isolates have been shown to be diazotrophic. Several strains are also chemolithotrophs, capable of using H 2 as energy and electron source and CO2 as carbon source. Besides assays for N2-fixing activity, DNA hybridisation to the well conserved molybdo-nitrogenase structural gene probe is an indicator of diazotrophy. However, absence of hybridisation to this probe, despite N 2 fixation activity, has been reported. Although the genetics of N 2 fixation in pseudomonads have hardly been studied, some nif genes have been shown to be plasmid-borne. Pseudomonas species are also predominant soil denitrifiers, reducing nitrate and nitrite to gaseous forms of nitrogen during anaerobic respiration. Hence, they play an important role in the global biological nitrogen cycle. Several diazotrophic species including a few pseudomonads can also denitrify. The potential contribution by Ne-fixing pseudomonads to the sinks and sources of soil nitrogen is considered small in the short term but essentially remains unclear in the absence of experimental data. Reliable rapid methods for their specific enumeration are indispensable for assessing their population dynamics and ascertaining their ecological significance.

Key words: Denitrification; H e oxidation; N 2 fixation; Pseudomonas species; Rhizosphere; Taxonomy

Introduction

N2-fixing organisms or diazotrophs are confined to the prokaryotes. Although not uniformly

* Corresponding author. Tel.: (613) 957 4347 Ext. 7530; Fax: (613) 992 7909; e-mail NUM347(a mbds.nrc.ca.

SSDI 01 6 8 - 6 4 4 5 ( 9 3 ) E 0 0 9 2 - X

distributed within any one genus, diazotrophic strains commonly exist in the genera of both archaeobacteria (eqv. archaebacteria [1]) and eubacteria. Free-living diazotrophs excluding photosynthetic bacteria and cyanobacteria are mostly heterotrophs with aerobic, microaerobic or anaerobic respiration when fixing N 2. Because of the high energy demand for N 2 fixation, free-liv-

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ing diazotrophic activity in the natural environment is generally limited by the availability of utilisable carbon substrates [2-4]. Limitation of carbon or energy substrates and the imposition of nitrogen deficiency should then favour the occurrence of N S i x i n g strains of the genus Pseudomonas since its members are well-recognised for their extreme nutritional versatility. Yet, few natural diazotrophic pseudomonads have been reported. The notion of a genuine N2-fixing pseudomonad has not gained general acceptance because of previous taxonomic uncertainties. This primarily stems from the historically non-stringent definition of the Pseudomonas genus, resulting in the inclusion of a large and remarkably heterogeneous group of polarly flagellated Gram-negative bacilli [5-7]. Nevertheless, there is no apparent reason why diazotrophy should not occur in the pseudomonads [4]. From a new perspective on the current emerging taxonomy of the pseudomonads, we attempt to clarify the diazotrophic status and the potential roles of this unusual group of pseudomonads with reference to plant-soil systems. Some early works are cited for thoroughness and historical development on the present topic which has not been previously focussed upon. Specialised functions (H 2 oxidation and denitrification) considered ancillary to N 2 fixation are discussed and compared to related diazotrophs with similar features. Several reviews in a recent monograph provide excellent information on the phylogeny [8], physio-ecology [9,10] and genetic regulation [11] of many wellknown diazotrophs.

Current taxonomy of the genus Pseudomonas

Pseudomonas-like organisms form one of the most complex groups of Gram-negative bacteria [7] apparently due to incomplete generic definition [6]. As the type genus of the family Pseudomonadaceae, its phenotypically based early definition is similar to that of the family [7]. Bacteria which are aerobic, polarly flagellated, Gram-negative, non-spore-forming, rod-shaped, and which metabolise sugars oxidatively, but do not readily fit elsewhere, are often broadly classi-

fied as Pseudomonas species. This practice has led to the accumulation of a large number of bacteria in the genus without clear taxonomic status. Pseudomonas species are usually differentiated by their phenotypic characteristics [12] in conjunction with numerical data analysis [13]. Modern taxonomic methods that have phylogenetic implications are D N A - r R N A hybridisation [14-16], enzymological patterning of conserved biosynthetic pathways [17], quinone and fatty acid composition [18,19], and 16S rRNA sequence analysis [20]. Despite the advances in methods for species classification, a rapid technique for routine phenotypic differentiation at the generic level is still needed. Polyamine analysis has been shown to be a reliable chemotaxonomic tool in differentiating bacteria at the genus and species levels [21,22]. A computer-based technique using Fourier-transform to analyse infrared signals of bacteria offers a promising tool for rapid identification at the subspecies level [23]. Developments in molecular genetic techniques will also undoubtedly facilitate genotyping. Examples are the use of low-molecular-mass RNA profiling [24] and rRNA-derived nucleic acid probes [25] to identify pseudomonads based on phylogenetic grouping. The taxonomic classification of the genus Pseudomonas has been undergoing revision based on phylogenetic relationships [6,14,23]. According to D N A - r R N A hybridisation data, pseudomonads were divided into five rRNA homology groups 1 to V [16]. This grouping was adopted in Bergey's Manual of Systematic Bacteriology with the exclusion of groups IV and V which really contained species of uncertain affiliation [7]. Using the same molecular technique on a large number of strains, De Vos and associates [6,15] concluded that the five rRNA groups are only remotely interrelated and therefore cannot be maintained in a single genus. They proposed that most Pseudomonas species are located on three rRNA branches and, therefore, divided into at least three genera: (a) the P. fluorescens branch consisting mostly of pigment-producing pseudomonads (equivalent to Palleroni's rRNA group I); (b) the P. acidouorans branch comprising most H 2oxidising pseudomonads (equivalent to Palleroni's rRNA group liD; and (c) the P. solanaceamm

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branch containing some phytopathogenic pseudomonads (equivalent to Palleroni's r R N A group II), Only members of the P. fluorescens rRNA branch (rRNA group I), with P. aeruginosa as the type species, are considered to be genuine pseudomonads (belonging to the genus Pseudomonas sensu stricto) while all other species were deemed generically misnamed [15]. In the rRNA homology group III sensu Palleroni [7], for instance, the genus Comamonas was proposed to accommodate P. acidocorans and P. testosteroni [26,27]. Willems et al. [28] proposed the genus Hydrogenophaga for the H2-oxidising Pseudomonas species of the same group which includes P.

)qava, P. palleroni, P. pseudoflat,a, P. carboxydoflava and P. taeniospiralis while Acidot,orax was suggested as the new genus for the remaining members (P. facilis and P. delafi'eldii) [29]. The taxonomy of P. saccharophila is still unclear. It has been placed close to the genus Leptothrix based on rRNA cistron similarity [14]. P. maltophifia, which represents Palleroni's rRNA group V, has been transferred to the genus Xanthomonas [30], Even rRNA group I will be subjected to further internal splitting if the latter is redefined by a 50% D N A - r R N A denaturation temperature difference (ATm~e)) o f _< 6°C from that of the type strain of P. fluorescens and by other chemotaxonomic markers [14]. However, currently valid species in Palleroni's rRNA groups II and IV still remain as members of the genus Pseudomonas despite their distant relationship with the phylogenetic nucleus of the newly defined authentic Pseudomonas genus. Hence, in the light of modern bacterial taxonomy based on rRNA cistron similarities, a phylogenetically defined family Pseudomonadaceae would be difficult if not impossible to maintain. The evolution of concepts on the pseudomonads regarding the development of their taxonomy is summarized in Table 1. Based on phylogenetic interpretations derived from the study of 16S rRNA sequences, the new class Proteobacteria was proposed to incorporate the "purple bacteria and their relatives" [20,31]. Four subdivisions, viz. alpha, beta, gamma and delta (with correspondence to earlier groupings called rRNA superfamilies I to IV [14]), were

Table 1 Evolution of concepts on the pseudomonads 1. P s e u d o m o n a d s in the b r o a d e s t s e n s e

Largely phenotypically defined Gram-negative, polarly flagellated rods with oxidative metabolism: hence, including many generically misnamed species lI. Pseudomonas rRNA homology groups 1, II and IV s e n s u Palleroni [6,15] With the above phenotypic characteristics and still valid species names (although these groups are only remotely related), including saprophytic or opportunistic pathogens. Ill. A u t h e n t i c g e n u s Pseudomonas s e n s u stricto Palleroni's rRNA group I [14,23]; phylogenelically defined genus with ubiquinone Q-9 as a proposed chemotaxonomic marker

placed under the new class. The P. acidot,orans (Palleroni's group III) and P. solanacearurn (Palleroni's group II) rRNA branches were classified under subdivision beta (rRNA superfamily II1) while the P. fluorescens branch (Palleroni's group I) was placed under subdivision gamma (rRNA superfamily II) [31]. D N A - r R N A hybridisation data of the unclassified saprophytic Pseudomonas species showed that two-thirds of them have been misclassified and are distributed in at least seven genera throughout the Proteobacteria [6]. However, for practical and other taxonomic reasons, thorough analyses of phenotypic differences among members of the genus Pseudomonas remain necessary before creating new species or genera [23]. Presently, there is no consensus in adopting molecular phylogenetic data as the only basis for the definition of genera.

Putative Nz-fixing pseudomonads Putative Ne-fixing pseudomonad strains have been identified as belonging to eight acknowledged Pseudomonas species (P. diminuta, P. fluorescens, P. paucimobilis (proposed new name Sphingornonas paucimobilis [32]), P. pseudoflat;a, P. putida, P. saccharophila, P. stutzeri and P. t:esicularis) listed in Bergey's Manual of Systematic Bacteriology and fall into the rRNA groups I, III

98

and IV [7] (Table 2). Of these, P. fluorescens [33], P. putida [34,35], and P. stutzeri strains JM300 [36,37] and CMT.9.A [36,38] belong to approved species in rRNA homology group I, which is kept in the genuine Pseudomonas genus classified under the Proteobacteria gamma subdivision (rRNA superfamily II) according to molecular phylogeny [6,14,23]. Further taxonomic studies of these strains, especially those including D N A - r R N A hybridisation, are obviously needed to ascertain their classification. Because of the heterogeneity of the non-fluorescent P. stutzeri species [23], the stability of its nomenclature may still be doubted by the taxonomic purist. Pseudomonas species strain 4B (Table 2) was originally identified as similar to P. delafieldii based on biochemical tests [39]. Its taxonomic status, however, remains elusive based on D N A - r R N A hybridisation data (J. De Ley, personal communication). On a similar basis, 'P. diazotrophicus' type strain H8 has

been determined as closely related to the Rhizobium-Sinorhizobium-Agrobacterium cluster 1 in the Proteobacteria alpha subdivision (rRNA superfamily IV) [14] and, therefore, does not belong to the genus Pseudomonas. Also misclassified is the N2-fixing 'P. rubrisubalbicans' [220], a pathogen of certain grasses. It is physiologically and taxonomically very similar to Herbaspirillum seropedicae [220-222], both belonging to a single rRNA sub-branch in the Proteobacteria beta subdivision (rRNA superfamily III). For convenience in these discussions, a putative N S i x i n g pseudomonad refers to a bacterium that has been: (a) identified as a member of the genus Pseudomonas according to the original genus definition, or reported as belonging to an acknowledged Pseudomonas species listed in Bergey's Manual of Systematic Bacteriology [7], and not disproved as a case of misclassification; and (b) tested positive for nitrogenase activity in a nitro-

Table 2 Reported N2-fixing pseudomonads Isolate or strain ~'

Habitat

Listed in BMSB h

Remarks [Ref.]

"Pseudomonas' spp.

Soil, water

* Pseudomonas 4B (ATCC 43/)38) d

Forest soil

No No

Unconfirmed [206-208] New strain; FFN "; n i f H D K present; incorporates

* Pseudomonas DC

Grass roots

No

Thermal spring

Soil

No No No No

"P. diazotrophicus" H8 (ATCC 35402) "alpha"

Rice roots

No

* P. diminuta "alpha"

Rice roots

Yes

* P. ambigua "P. azotocolligans" (NC1B 9391)"alpha' I "P. azotogensis' (NCIB 9277) "P. azotogensis" strain v

Soil Soil

15N2; oxidises H 2 [36,39,63,98] New strain; FTN; fluorescent; nifHDK present: psychrotrophic; oxidises H ~; denitrifies [36,39,47,48,98,114] VFN [209] Proved to be non-diazotrophic; mixed culture [66,210] Proved to be non-diazotrophic [51,66,210-213] Incorporates 15N2; misclassified [66,210-213] Type strain of new species; considered to be in r R N A superfamily IV close to dgrobacterium [14]; oxidises H 2; denitrifies; nifl-lDK present; misclassified [14,39,46,150,193] New strain; considered to be in r R N A superfamily IV and not an authentic Pseudomonas [14];

FTN [52] * ~ P. fluorescens 'gamma' Soil **1~ fluorescens var. indologenes, nov. var. Acidic fl)rest soil . t p. fluorescens var. t'allis urnbrosae, nov. var. Acidic forest soil "P. glathei' (ATCC 29195) Acid laterite "P. methanica' Pond mud, soil "P. methanitri-ficans" Freshwater~ soil

Yes Yes

Genetically constructed from a laboratory strain [68] New (unrecognised) variety; FTN; phychrotrophic [33]

Yes Yes

New (unrecognised) variety; FTN; psychrotrophic [33] Type strain; non-diazotrophic [214] Unconfirmed [70,215] Contaminated with Methylosinus [216,217]

No No

99 Table 2 (continued) Isolate or strain ~

Habitat

Listed Remarks [Ref.] in BMSB b

* P. paucimobilis 5AJ 'alpha'

Rice root

Yes

* P. pseudoflaca 'beta'

Freshwater

Yes

** P. putida MT20-3 'gamma'

Yes

** P. putida 'P. rubrisubalbicans' ' beta'

Arctic grass roots Yes Sugar cane, Yes grass leaves

* P. saccarophila (ATCC 15946)"beta'

Pond mud

Yes

** P. stutzeri JM300 "gamma'

Soil

Yes

, t p. stutzeri CMT.9.A

Sorghum roots

Yes

* P. t,esicularis 'alpha'

Rice roots

Yes

New strain; considered to be in rRNA superfamily IV close to Zymomonas [14]; FTN [46,53,218]; proposed new genus Sphingomonas [32] New strains; FTN; nifHDK present; oxidise H 2 [92]; new genus Hydrogenophaga [28] Genetically constructed from a modified laboratory strain [69] New strain; FTN; psychrotrophic; oxidise H 2 [34-36] Considered to be in rRNA superfamily llI similar to Herba~pirillum; FTN; phytopathogen; incorporates 15N2; misclassified [14,220-222] Approved type strain; considered to be in rRNA superfamily Itl and not an authentic Pseudomonas [14,224] incorporates 15N2; oxidises H 2 [116,148] Approved strain: nifDK absent: oxidises H2; denitrifies [36,37,98] New strain; FTN; nifDK absent; incorporates tSN2; oxidises H 2 [36,38,98] New strain; considered lo be in rRNA superfamily IV and not an authentic Pseudomonas [14]; FTN [52]

Putative N2-fixing Pseudomonas species strains (indicated by asterisks) are defined as strains identified as belonging to the genus Pseudomonas, or to a recognised Pseudomonas species [7], and tested positive for nitrogenase activity. Names of non-putative ones are enclosed within quotation marks (see text text for details). An organism considered to be a member of the authentic Pseudomonas genus [14] is indicated by a dagger (t). '~ Most of the strains were tested by the acetylene reduction technique. b Palleroni's contribution to Bergey's Manual of Systematic Bacteriology [7]. " Further tests needed are DNA-rRNA homology or other molecular biological and biochemical tests. a American Type Culture Collection, Rockville, MD. National Collection of Industrial Bacteria, Aberdeen, Scotland, UK. Proteobacteria subclass according to De Vos et al. [6] and Stackebrandt et al. [31].

g e n - d e f i c i e n t m e d i u m . N a m e s o f r e p o r t e d isolates o r s t r a i n s t h a t a r e n o t c o n s i d e r e d p u t a t i v e N2-fixing p s e u d o m o n a d s by t h e s e c r i t e r i a a r e u s e d w i t h i n q u o t a t i o n m a r k s , T h i r t e e n p u t a t i v e N2-fixing p s e u d o m o n a d s a r e i n d i c a t e d in T a b l e 2. Interestingly, the genera Azotobacter and Azomonas, which contain active aerobic hete r o t r o p h i c N2-fixing s p e c i e s , b e a r c l o s e p h y l o g e netic relationship with the authentic Pseudomonas genus although they are morphologically d i f f e r e n t f r o m t h e p s e u d o m o n a d s . T h e i r s i m i l a r i t y is s u p p o r t e d by r R N A analysis, a m i n o a c i d s e q u e n c e o f c y t o c h r o m e Cs51 a n d t h e c o n s e r v a t i o n o f o t h e r g e n e s o r g e n e p r o d u c t s [14]. T h e latter include DNA sequence homology of nil structural genes with the well-known diazotrophic g e n u s Klebsiella, w h i c h also b e l o n g s to t h e P r o -

teobacteria subdivision gamma (rRNA superfamily II).

General ecology The nutritional and catabolic versatility of P s e u d o m o n a s s p e c i e s u n d o u b t e d l y c o n t r i b u t e s to t h e i r u b i q u i t y in n a t u r e . B e c a u s e o f this a d v a n t a g e , e s p e c i a l l y in t h e i r a e r o b i c c o m p e t i t i o n for c a r b o n [40], p s e u d o m o n a d s c a n b e f o u n d in vario u s h a b i t a t s (e.g. soil, w a t e r , h u m a n s , a n i m a l s , plants, a n d f o o d s ) w h e r e t h e y c a n be s a p r o p h y t i c a n d m a y b e c o m e b e n e f i c i a l o r p a t h o g e n i c to t h e i r h o s t s [7,12,41,42]. T h e y a r e o n e o f t h e d o m i n a n t g r o u p s o f b a c t e r i a in m a r i n e [43] a n d soil [44] systems including the rhizosphere where they can

I (1(I

fix N 2 [35,38,45-53], produce plant growth-promoting substances [34,54,55] or excrete siderophores that exert antagonistic activity against soil-borne pathogens [56,57]. Hence, they have been considered as potential biological fertilizers and pesticides [34,58-61]. However, such pseudomonads may stimulate plant growth without significantly contributing to plant nitrogen as in rhizocoenoses involving azotobacters or azospirilla [3]. This may be due to their indirect nitrogen contribution which occurs mainly after death and mineralisation of their cellular nitrogenous components. Alternatively, the plant growth-stimulating effect may be more pronounced than N 2fixing activity in pseudomonads that have both properties, e.g.P, putida GRI2-2 [34,35]. Geo-

graphically, pseudomonads can be found in the tropical [7], temperate [47,48], arctic [35] and antarctic [62] regions. Free-living diazotrophs are considered to contribute significantly to the nitrogen economy of highly productive ecosystems such as marshlands or those with high C:N ratio [2,3]. They may also be synergistically active in degrading recalcitrant substances. An example of the pseudomonads with such capabilities is Pseudomonas species 4B which was isolated by enrichment with benzoate [39,63]. It was found to have high N 2 fixation activity in co-culture with a cellulolytic bacterium, Cellulomonas sp. CS1-17, using plant residues or their degradation products as carbon substrates

[64,65] (Table 3).

Table 3 M a x i m u m N 2 fixation rates reported fl)r P s e u d o m o n a s

System

systems

Method

Assay conditions ~'

Reported rate [Ref.]

25-ml L C b

Kjeldahl

Aerobic, 30°C

100-ml L C

Kjeldahl

Aerobic, 30°C ' A n a e r o b i c ' , 30°C

Concentrated

~SN

0.55 mg N 14 days l [208]; 5.6 mg N 5 w e e k s 50/xgNml t4days l 40 Izg N ml " i 4 days > 0.1 at.% exc. 15N 4 h [207]

' P s e u d o m o n a s ' spp.

cell s u s p e n s i o n

Aerobic or 'anaerobic', 30°C

i [207]

* P s e u d o m o n a s 4B

tATCC 43038) 10-ml LC 10-ml LC 12-ml SS c co-culture d

Microaerobic, 28°C

C2H 2 15N C2H :

do.

Aerobic, 28°C

1 . 6 / x m o l C 2 H 4 mg 1 protein h I 0 . 5 2 / z m o l N 2 fixed mg - 1 p r o t e i n h - t [39] 12.7 txmol C 2 H 4 d a y - I [65]

5-ml SS culture

(?2 H 2

Microaerobic, 28°C

0.95 izmol C 2 H 4 mg

D e s c h a m p s i a roots (habitat of D C )

C2H 2

Microaerobic, 22°C

0.25+_0.29 tzmol C 2 H 4 g

Kjeldahl

Aerobic, 30°C

1.93 mg N 3 0 - 4 5 days

C2H 2

C2H 2

Aerobic, 32°( ` M i c r o a e r o b i c , 3I)°C,

18 nmol C 2 H 4 h z [150] 22 n m o l C 2 1 t 4 mg I protein h

C 2H z

chemolithotrophic A e r o b i c , 24-h field assay

21) ~ m o l C 2 H 4 hill - t day

Kjeldahl

Aerobic, 26°C

0.9mgN

* P s e u d o m o n a s DC

t p r o t e i n h - t [47,48] i dryweight h

* P. a m b i g u a

30-ml LC

t [209]

P. diazotrophicus'

H8 (ATCC 35402) 3-ml SS culture 10-mL L C

Wetland rice plants (habitat of H8)

I [116]

i [135]

* P. f l u o r e s c e n s ~

20-ml L C

100ml

t 15 days

1133]

~ [219]

101 Table 3 (continued) System

Method

Assay conditions a

20-ml LC

C2H 2

Microaerobic, 27°C, chemolithotrophic 39.1 nmol C2H 4 mg t protein h -~ t [92]

* P. putida GRI2-2 5-ml SS culture

C2H 2

Aerobic, 14°C

17.4 nmol C2H 4 h 1 [34,35]

tSN C2H 2 C2H 2

Microaerobic, 28°C Microaerobic, 30°C do., chemolithotrophic

6.3_+1.9 at.% exc. lSNday l [116] 2.4/zmol C2H 4 m g - 1 protein h - 1 0.52#mo1C2I-t 4 mg 1 protein h-1[148]

C2H 2 15N

Microaerobic, 28°C do.

0.8/2mol C2H 4 m g - I protein h i [38] 2.1 /zmol N 2 fixed mg t protein 15 days- i [38] t

* P. pseudoflava (NEU

* P.

Reported rate [Ref.]

2226)

saccharophila

(ATCC 15946) 20-ml SS culture 2-ml LC 2-ml LC * P. stutzeri CMT.9.A

Cell suspension 600-ml LC

The rates compiled are for heterotrophic N 2 fixation unless otherwise stated. Conversion to standard rate units is not always possible because of insufficient data from most of the cited references. Putative Nz-fixing pseudomonads (cf. Table 2 footnotes for definition) are indicated by asterisks. See references for details. h Liquid culture. c Semisolid agar. d Co-culture with a Cellulomonas sp. in the presence of wheat straw. lsolates from forest soils. f Recalculated from data in Krotzky and Werner [38].

Diazotrophy According to Palleroni [7], pseudomonads lack N2-fixing ability. The ability of the pseudomonads to fix atmospheric N 2 has not been appreciated [7,66,67] usually because of the following problems [39]: (a) mistaken or uncertain identification of the diazotroph; (b) lack of rigorous tests for diazotrophy in pseudomonads; and (c) multigeneric nature of the genus Pseudomonas as discussed above. Early attempts to find an N2-fixing pseudomonad were unsuccessful, due to the failure to recognise the 02 sensitivity or microaerobic character of its N2-fixing system. The existence of putative diazotrophic pseudomonads is now substantiated. Among these, Pseudomonas species strains 4B, DC and P. pseudoflava have been demonstrated to possess nil structural genes while Pseudomonas sp. 4B, P. saccharophila and P. stutzeri CMT.9.A have been shown to incorporate ~SN2 (Table 3). The genetically constructed N2-fixing strains of P. fluorescens and P. putida indicate that these aerobic species have mecha-

nisms to accommodate and express nil genes from Klebsiella pneumoniae although their maintenance may be a problem in the absence of selection pressure and protection from 0 2 repression or inhibition [68,69]. The contribution by free-living or plant-associated heterotrophic diazotrophs to soil nitrogen in agriculture is small relative to that by legume symbiotic systems [3]. Specific estimations of the potential contribution of putative diazotrophic pseudomonads in the rhizosphere or natural environments have not been reported. Generally, their free-living N 2 fixation rates are low with the exceptions of those with rates in the same order of magnitude as that of Azospirillum lipoferum [48], viz. Pseudomonas sp. strains 4B, DC, P. saccharophila and, possibly, P. stutzeri CMT.9.A (Table 3). In the case of P. saccharophila, its chemolithotrophic N 2 fixation rate is lower than that under heterotrophic conditions. It should be noted that the reported rates are probably underestimations of potential rates since they were usually not determined under well-defined or op-

102 timal conditions, which were often unknown or not investigated. Moreover, because of the different methods used in N 2 fixation assays (see below), direct comparisons of the reported rates cannot be readily made.

Determinative tests

Tests for diazotrophy can be grouped into three types: cultural, analytical, and genetic. Experimentation on the combination of optimum growth and assay conditions is necessary in the search for low-activity diazotrophs such as pseudomonads. The merits of each type of test and caveats in interpreting the results are discussed below. Cultural tests. The cultural test is the earliest one used to isolate diazotrophs. In the past, growth of an organism on a nitrogen-deficient medium would usually be interpreted as due to N x fixation. Cultures on 'N-free' (strictly speaking N-deficient) media incubated in the presence or absence of air have been the experimental conditions used to isolate azotobacters, beijerinckias and clostridia. U n d e r such conditions, bacteria that fix N 2 microaerobically and prefer N-deficient media containing 'starter' nitrogen are likely to be missed. On the other hand, organisms which have the ability to scavenge traces of volatile nitrogenous compounds such as ammonia from the immediate atmosphere or organisms which could assimilate contaminating nitrogenous compounds in the medium could be misconstrued as Nz-fixing [66]. The test, however, is still useful in isolating putative N 2 fixers. A modification of the solid 'N-free' medium is the 'N-free' semisolid agar medium contained in a culture tube to provide a gradient of 0 2 regimes: high at the surface and low at the bottom [70,71]. A small amount of yeast extract is incorporated into the semisolid medium to supply organic growth factors and 'starter' nitrogen which promotes growth of the Nx-fixing bacteria but does not inhibit acetylene reduction [72-74]. A single carbon source or a mixture of carbon sources that approximate the natural niche is incorporated into the medium [73-75]. This isolation procedure in combination with the acetylene reduction test has yielded many different kinds of aerobic heterotrophic dia-

zotrophs such as azospirilla, bacilli, enterobacters and pseudomonads from various ecosystems. Recently, various enrichment or enumeration methods for most free-living diazotrophs have been detailed by Knowles and Barraquio [76]. Analytical tests. Analytical tests used to detect diazotrophy measure directly or indirectly either substrate consumption or product formation of the nitrogenase reaction [77-80]. An earlier direct technique to determine the Nz-fixing ability of an organism is measuring the increase in dry weight or total Kjeldahl nitrogen content of the culture in an initially 'N-free' medium. The technique suffers the same disadvantages as growth on solid 'N-free' media and is very slow and insensitive. The acetylene reduction technique [81,82] is the most rapid and sensitive analytical assay that has been used routinely for measuring nitrogenase activity of pure cultures, cell-free extracts, purified enzymes and natural samples. Since it is indirect, acetylene reduction rates must be calibrated (by ~SN enrichment or dilution) with N 2 fixation rates under identical conditions to obtain an accurate conversion ratio between the two substrates if absolute quantitation of N 2 reduction is required. Acetylene reduction activity is usually indicative of N 2 fixation potential, even for a complex ecosystem providing the assay is properly carried out to avoid erroneous results caused by experimental [83-85] or potential [86] problems associated with the use of acetylene. A T P - d e p e n d e n t H 2 evolution is a by-product of the nitrogenase reaction and its measurement in the presence of acetylene to prevent uptake hydrogenase activity [87] may be used as an indirect estimate of N 2 fixation in the absence of any substrate other than endogenous protons. The technique would be more useful for assaying alternative nitrogenase systems which evolve H E at high rates [88]. The only unequivocal demonstration of diazotrophy is the determination of 15N2 incorporation into bacterial cells. Postgate [89] stated that, to be convincing, the report of a new N2-fixing system must fulfill two elementary criteria: it must be unequivocally free of known diazotrophs and must demonstrate significant uptake of ~SN2. This

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test, however, is not as sensitive as the acetylene reduction assay and requires access to either a mass spectrometer or an emission spectrometer. Among the putative N2-fixing pseudomonads, Pseudomonas sp. 4B, P. saccharophila and P. stutzeri CMT.9.A were reported to have significant 15N2-fixing activity (Table 3). Genetic tests. The structural genes for molybdo(Mo)-nitrogenase are highly conserved among N2-fixing organisms [90]. Hence, Southern hybridisation [91], in which the Mo-nitrogenase structural genes nifHDK (usually obtained from the well-studied diazotroph Klebsiella pneumoniae M5AI, eqv. K. oxytoca) are used to probe an organism's genomic DNA, has been employed as a test for diazotrophy [39,90,92-97]. Strictly, such probing is a test complementary to activity assay for indicating the genetic similarity between nitrogenases from different species. This technique could be helpful when the nil gene expression of a strain is not detectable by activity assay [92], due to inappropriate assay conditions or genetic defects. However, since diazotrophy is more appropriately defined by phenotype rather than genotype, an organism with defective or nonfunctional nif genes cannot actually be considered an N 2 fixer. Despite the high interspecies homology of Mo-nitrogenase genes [90], Fallik et al. [98] did not find hybridisation at moderate stringency with the genomic DNA of acetylenereducing P. stutzeri strains CMT.9.A and JM300 by using a nifDK probe encoding the large subunits of Mo-nitrogenase from Azotobacter chroococcum. Further hybridisation using alternative nitrogenase gene probes (t,nfDGK and anfDGK ) from A. chroococcum and A. L,inelandii also failed to detect vanado- or iron-only nitrogenases. Since the nif genes of P. stutzeri CMT.9.A are plasmid-borne [38], they could have been lost through plasmid curing during routine subculturing. Plasmid-borne nif genes are known in diazotrophs of the genera Desulfol:ibrio [96], Enterobacter [99], Lignobacter [100], Rahnella [101] and Rhizobium [102]. As for JM300, the genomic location of its nif genes has not been investigated and its diazotrophy has yet to be confirmed by 15N2 incorporation. It also remains possible, however, that the nitrogenase of these pseudomonads

is genetically not closely related to the azotobacter enzymes. Homology with Mo-nitrogenase structural genes has been detected in the genomes of the putative pseudomonads Pseudomonas sp. strains 4B and DC, and several P. pseudoflat,a strains (Table 2). Besides P. stutzeri strains CMT.9.A and JM300, diazotrophs which lack DNA homology with nif probes have been reported, e.g. Methylosinus sp. [94], two strains of Agrobacterium tumefaciens [103,104] (see also below) and the autotrophic thermophile Streptomyces thermoautotrophicus UBT1 [105]. Hence, diazotrophy may not be determined solely from hybridisation results with gene probes from limited sources. To date, alternative nitrogenase gene-like sequences have not been reported in pseudomonads. Finally, a note of caution is needed on using nitrogenase gene probes that include DNA encoding the small subunits (nifH, t,nfH or anfH) for diazotrophy assay. Fallik et al. [98] avoided their use in the detection of nitrogenases largely because of the possible lack of specific involvement in N 2 fixation despite homologous sequences. For example, nifH-like sequences have been located in a photosynthetic gene cluster in Rhodobacter capsulatus [106], the chloroplast genome of the liverwort Marchantia polymorpha [107] and the N2-fixing cyanobacterium Plectonema boryanum [108]. Recently, a nifDK-like gene in the P. boryanum chloroplast was also found [109], indicating that some chloroplast proteins and nitrogenase could have evolved from a common ancestral origin.

Limiting factors The agricultural significance of heterotrophic N 2 fixation by free-living bacteria is generally viewed as small and variable in the short term since it is subject to environmental conditions. Exceptions noted above are highly productive ecosystems with high C:N ratio [2,3]. The contribution of free-living diazotrophs to plant nitrogen is considered limited and indirect because they may release nitrogenous compounds only after cellular disintegration and mineralisation. Hence, to enhance N 2 fixation by these agents, it is important to understand the factors and underly-

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ing physiology that limit indigenous nitrogenase activity before attempting to minimize their effects by implementing appropriate farm management practices. Results from ecological and physiological studies on Nz-fixing pseudomonads are insufficient for extrapolation to natural environments. Relevant information is therefore largely drawn from other aerobic heterotrophic N2-fixing systems [110-113] in the following discussion. It must be pointed out that a single limiting factor may dominate a specific environment only periodically. Co-existing factors in nature usually interact with each other to control indigenous nitrogenase activity. Energy and reducing sources. Large amounts of energy in the form of ATP and reducing equivalents are required for the N 2 fixation reaction. These requirements are normally provided by organic carbon compounds. A heterotrophic aerobic diazotroph such as Azotobacter species not only must compete with non-diazotrophs for these sources, it must also allocate them for functions supportive of, or ancillary to, N 2 fixation, e.g. respiratory protection against 0 2 damage to nitrogenase and assimilation of ammonia, the nitrogenase reaction product. Unless considerable amounts of oxidisable carbon substrates are available, N 2 fixation by free-living or associative bacteria will always be insignificant. However, most aerobic diazotrophs lack elaborate respiratory protection and require 0 2 for their oxidative metabolism. A common strategy used to approach this problem is to restrict 0 2 diffusion by cell clustering which has been observed in Pseudomonas species DC [114]. Since they are microaerophilic during Nz-fixing growth [115] (see below), maximum energy demand for protection against 0 2 as in azotobacters is not needed. A wide range of aliphatic and cyclic carbon compounds can be utilised as carbon and energy sources by pseudomonads [12]. These include simple sugars, polysaccharides, organic acids, polyols, amino acids, hydrocarbons and aromatic compounds. They are expected, and in some cases shown, to support nitrogenase activity [38,39,46, 48,63,92,116] as well as saprophytic competitiveness. Interestingly, the well-studied aromatic degradation pathways of pseudomonads also exist

in N2-fixing azotobacters [117,118] which are phylogenetically closely related to the authentic Pseudomonas species [14]. The fact that addition of metabolisable organic substances increases nitrogenase activity in soils or soil-plant systems suggests that these compounds are limiting in the natural environment. In field conditions, the supply of organic substances comes from living roots, sloughed-off cell debris or crop residues. Straw incorporated into soil usually increases its total nitrogen content, presumably by increasing the C:N ratio and, thus, indigenous N 2 fixation activity. As mentioned above, Pseudomonas sp. 4B could utilize wheat straw and its degradation products to support nitrogenase activity in pure culture or co-culture with a Cellulomonas species (Table 3) [64,65,119]. In addition to straw breakdown products, straw components such as xylan can be utilised directly by some free-living N2-fixing bacteria as energy sources [120-122]. A wide range of direct and indirect data has indicated that photosynthates are the main carbon and energy sources supporting associative or symbiotic N e fixation [123-126]. Combined nitrogen. In all free-living aerobic diazotrophs, ammonium represses synthesis and inhibits the activity of nitrogenase [127]. Free-living and associative Nz-fixing bacteria respond differently to combined nitrogen but it is well established that assimilable combined nitrogen is preferred o v e r N 2 as a nitrogen source. N z fixation will take place only when the level of available combined nitrogen is below a certain threshold value, which differs for various organisms and under different circumstances. In water culture, ammonium at 0.33 mM depressed N 2 fixation associated with wetland rice [128]. However, Watanabe et al. [129] showed no appreciable difference in field measurements of N 2 fixation in the soil alone or in wetland rice plots with or without nitrogen fertilizer treatment. Gilmour et al. [130] reported that the application of mid-season nitrogen fertilizer (45 kg N ha ~) to rice plants caused an initial depression in nitrogenase activity of the rice root system followed by an enhancement in nitrogenase activity. Some tropical grasses grown in the field and assayed 2 weeks after top dressings of 20 kg N ha-~ were not

105 affected in their rhizospheric nitrogenase activity [131]. No effect was observed even after eight similar dressings. Using a hydroponic technique, Zuberer and Alexander [132] showed that the presence of 10-20 ppm ammonium-N reduced root-associated acetylene reduction activity in Zea mays and other gramineous species by 75-90% within 24 h of addition. However, acetylene reduction activity resumed as ammonium became depleted in the solution. All these observations on plant systems can be explained by the removal of combined nitrogen as an inhibitor of nitrogenase via plant uptake, assimilation by microflora or adsorption to soil. Small amounts of ammonia or amino acids as 'starter' nitrogen, in fact, could stimulate N 2 fixation and growth of diazotrophs associated with wetland rice roots [74]. In semisolid agar with a combined carbon source, Pseudomonas sp. 4B showed higher nitrogenase activity in the presence of inorganic nitrogen [119]. It is apparent that small doses of combined nitrogen either do not affect or have some stimulatory effect on N 2 fixation, indicating the possibility that N 2 and fertilizer nitrogen can be utilised simultaneously. Gibson et al. [119] suggested that if a low level of nitrogen can be applied at inoculation with free-living diazotrophs, it would hasten straw degradation without inhibiting N 2 fixation even if nitrogenase activity is not stimulated. Inhibition by 0 2. Nitrogenase is O2-sensitive. Its synthesis and activity are respectively repressed and inhibited by 0 2 [115,127,133]. N 2 fixation by free-living aerobic heterotrophs occurs under microaerobic to aerobic conditions. Like most aerobic N 2 fixers, diazotrophic pseudomonads are microaerophilic when fixing N 2 [38,39,46, 47,63,92,114,116,134]. In natural environments, soil moisture may indirectly influence nitrogenase activity by affecting 0 2 movement into the soil pores. When soil moisture is increased, 0 2 diffusion is retarded. Nitrogenase activity of strawamended sand cultures of Pseudomonas sp. 4B and other free-living bacteria increased with increasing moisture levels [119]. N2-fixing pseudomonads were not found in the roots of rice and other plants grown under dryland conditions [135,136]. This seems to be consistent with the finding that N 2 fixation associated with wetland

plants is much higher than that of dryland plants [135,137,138]. Conceivably, wetland habitats may be low in available nitrogen due to high rates of denitrification and leaching of nitrates [137], or there may be a higher number of N2-fixing bacteria associated with wetland plants due to their selective development [135]. Conversely, dryland condition is too aerobic to select for N2-fixing pseudomonads. Zuberer and Alexander [132] reported high acetylene reduction rates associated with intact roots of Zea mays and other gramineous species in hydroponic culture exposed to reduced 0 2 tension (2% v / v O2), whereas little or no activity was observed in roots exposed to N 2 or air, respectively. These results were subsequently confirmed by 15N2 incorporation data [139]. Other encironmental factors. A near neutral pH environment is expected to be optimal for N 2 fixation by pseudomonads from studies of pure cultures [12,39,46,48,116]. Other aerobic heterotrophic organisms show a wide pH tolerance [111]. A bacterium which exhibits nitrogenase activity at pH below 3.0 has been reported [140,141]. N2-fixing 'P. diazotrophicus' H8 was found predominant in the roots of wetland rice and water hyacinth grown in Maahas soil (pH 6.7) in the Philippines [46,74,136]. Its occurrence in acidic soils in the Philippines is doubtful [142]. O'Toole and Knowles [143] found that high concentrations of glucose under aerobic conditions suppressed nitrogenase activity and they concluded that this effect was caused by the lowering of pH from the original 6.8-5.6. Roper and Smith [144] observed that N 2 fixation by free-living bacteria using straw as an energy source was most efficient in soils with a neutral pH and a high clay content, provided moisture and temperature were adequate. Diazotrophs are mostly mesophilic. Although some diazotrophic cyanobacteria are thermophilic, only one actinomycete (Streptomyces thermoautotrophicus) [105] and one archaeobacterium ( Methanococcus thermolithotrophicus ) [223] are able to fix N 2 actively above 60°C. However, no true psychrophiles have been discovered. A few rhizobia [145] and diazotrophic pseudomonads [33,35,114] have been reported to be psychrotrophic. The N2-fixing activities of these or-

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ganisms are not only limited by low temperature itself but also by the increasing dissolved O, with decreasing temperature. Non-N2-fixing microorganisms are expected to affect N 2 fixation rates and availability of the fixed nitrogen to plants via synergistic or antagonistic relationships. This aspect of microbial interactions has already been addressed by Jagnow [146] and Vose [147].

H, oxidation and chemolithotrophy The ability to oxidise H 2 is a unique property of 'hydrogen (Knallgas) bacteria'. Most of the members in Pseudomonas rRNA homology group Ill sensu Palleroni ( i . e . P . facilis, P. saccharophila, P. Jlal~a, P. pseudoflat,a, P. carboxydoflaca, P. palleroni and P. taeniospiralis) are 'hydrogen pseudomonads' [7,23]. With the exeption of P. saccharophila, they have been assigned to the genera Acidororax and Hydrogenophaga (see above). P. saccharophila [116,148] and some strains of P. pseudoflaca [92] are diazotrophic (Table 2). Other N S i x i n g pseudomonads with detectable H2-oxidising activity are Pseudomonas sp. str. 4B and DC, 'P. diazotrophicus' H8, P. putida G R I 2 - 2 , and P. stutzeri JM300 [36,39,46, 148-150]. Among them, the relation between H 2 oxidation and N 2 fixation in P. saccharophila is the best studied [148]. ATP-dependent H 2 evolution by nitrogenase is a consequence of proton reduction concomitant with N 2 reduction to ammonia. In the presence of O , as electron acceptor, this H~ is oxidised to water in most aerobic diazotrophs through the action of a membrane-bound, irreversible uptake type of hydrogenase. Uptake hydrogenase is present in almost all N2-fixing bacteria [151-154]. The physiological role of uptake hydrogenase in N S i x i n g bacteria has been hypothesized to recycle the H 2 evolved during N 2 fixation. Theoretically, N 2 fixation efficiency will increase in the presence of an active H 2 recycling system due to the following beneficial effects: (a) protection of nitrogenase from O 2 inactivation; (b) conservation of ATP or reducing power for nitrogenase reaction; and (c) protection of nitrogenase from H 2 inhibition [153,155]. Evidence supporting these benefits of uptake hydrogenase

for N 2 fixation has been observed: (a) H 2 oxidation via hydrogenase is coupled to the electron transport chain with O~ as the electron sink, thus keeping 0 2 away from the vicinity of nitrogenase. The experimental test for this is the shifting of the optimum nitrogenase activity from lower to higher 0 2 tension [153]. This phenomenon was observed in Azotobacter chroococcurn under carbon-limited conditions [156], but not in Azospirillure brasilense [157]. (b) The electrons generated by the uptake hydrogenase reaction pass through the electron transport chain and produce ATP (by oxidative phosphorylation) and reducing power [154]. This ATP may be utilised in the nitrogenase reaction. Walker and Yates [156] reported utilisation of H~ as electron donor but no H2-supported ATP generation in A. chroococcure. Fu and Knowles [158] observed the increase in acetylene reduction activity in the presence of H : in carbon-starved cells of Azospirillum lipoferum and A. amazonense. The ability of 'P. diazotrophicus' H8 [150], P. pseudoflat'a [92] and P. saccharophila [116,148,159] to grow under chemolithotrophic N2-fixing conditions suggests that H , oxidation via hydrogenase and the electron transport chain generates reducing power and ATP not only for N~ fixation but also for CO~ fixation. (c) H ~ is a competitive inhibitor of N~ reduction [160]. Protection of nitrogenase from H~ inhibition is considered by Mortenson [161] as a primary function of hydrogenase. This protective mechanism of hydrogenasc was demonstrated in Xanthobacter autotrophicus but only when H~ was exogenously provided, suggesting that the concentration of H~ evolved by nitrogenase was not inhibitory [162]. In Azotobacter species, the intracellular H 2 concentration also would not increase sufficiently to inhibit N, reduction [156]. The site in the respiratory chain where the electrons from H 2 e n t e r is not yet known. In autotrophically grown cells of P. saccharophila the electrons from H , were reported to entcr at cytochrome b bypassing NADH dehydrogenase [163-167]. Because specific inhibitors (rotenone and atebrin) of energy coupling Site I in the electron transport chain did not eliminate phosphorylation linked to H , , but eliminated NADH-

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associated A T P generation, it was concluded that only coupling Site II (between cytochromes b and c) was involved in H 2 oxidation. Site III was found to be non-functional. Donawa et al. [164] suspected, however, the existence of a phosphorylation (proton translocation) site between H 2 and cytochrome b to account for the ATP formed. There are suggestions that the entry site is at the ubiquinone level in some H2-oxidizing diazotrophs [168,169]. Generally, the synthesis and activity of hydrogenase in aerobic diazotrophs including free-living rhizobia is regulated by 0 2, H2, carbon substrates, nitrogen sources, and nickel. D N A topology has been suggested to regulate hydrogenase synthesis [170] based on the repression of hydrogenase expression when D N A gyrase inhibitors were added to the derepression medium [171]. 0 2 represses hydrogenase synthesis in Alcaligenes latus [172], Azospirillum brasilense [173,174] and Pseudomonas saccharophila [134]. Hydrogenase activities of A. brasilense [174] and A. lipoferum [173,175] are inhibited by 02, whereas those of A. amazonense [173] and P. saccharophila [134] are not. H 2 induces the synthesis of hydrogenase in A. latus [172], P. saccharophila [134] and X. autotrophicus [162], while that of hydrogenase in A. brasilense appears not inducible [174]. The H 2 evolved during N 2 fixation is most likely responsible for stimulating hydrogenase expression in A. latus [172] and P. saccharophila [134]. Enzyme synthesis is repressed by high concentrations of organic substrates also in these two species. Their nitrogenase and hydrogenase are, however, synthesized independently. Hydrogenase is expressed in NH~-containing medium in the presence of H 2 suggesting that NH]" per se does not inhibit hydrogenase activity. Many hydrogenases contain nickel [176]. Nickel-containing hydrogenases appear to be more tolerant to 0 2 than those without nickel [177,178]. The membranebound hydrogenase of P. saccharophila [179] possibly contains nickel based on its requirement for this metal during chemolithotrophic growth and hydrogenase synthesis [159]. Organisms that use CO 2 as the sole carbon source and reduced inorganic compounds such as H2, H2S, Fe 2+, NO 2 or NH 3 as energy and

electron source are chemolithoautotrophs [180]. The diazotrophic pseudomonads are Hz-oxidising facultative chemolithotrophs, i.e. organisms that can grow in an atmosphere of CO 2, H 2 and N 2 when organic carbon supply is limited. An N2-fixing chemolithotroph was first reported by Ooyama [181]. Later, Gogotov and Schlegel [182] described similar types of bacteria. Growth of a n H 2 chemolithotroph under N2-fixing conditions, therefore, requires three important enzymes: hydrogenase, ribulose 1,5-bisphosphate carboxylase and nitrogenase. Diazotrophic bacteria that can oxidize H 2 under Nz-fixing or non-N2-fixing conditions may be classified as follows: (a) facultative chemolithotrophic H 2 oxidisers which utilise H 2 mixotrophically (with organic carbon as the main carbon source) or autotrophically (with CO 2 as the alternative carbon source); (b) heterotrophic H 2 oxidisers which utilise H 2 only under heterotrophic conditions (with organic carbon as the carbon source); and (c) obligatory chemolithotrophic H 2 oxidisers. Azospirillum lipoferum, Derxia gummosa [183,184], Pseudomonas sp. DC [39], 'P. diazotrophicus' H8 [150], P. pseudoflaL~a [92], P. saccharophila [116,148,159] and Xanthobacter autotrophicus [162] are examples of facultative chemolithotrophic H2-oxidising diazotrophs [185,186]. Azomonas agilis, Azospirillurn

brasilense, Azotobacter t,inelandii, Bacillus polymyxa and Beijerinckia indica are N2-fixing heterotrophic H 2 oxidizers which do not have the ability to grow chemolithotrophically [152,185187]. Streptomyces therrnoautotrophicus oxidises H 2 and reduces CO or CO 2 while fixing N 2 aerobically and, therefore, is an obligate chemolithoautotroph [105]. The aerobic and autotrophic iron-oxidising Thiobacillus ferrooxidans which can fix N 2 [188,189] has been shown to contain strains that are capable of H 2 oxidation [190]. It is not yet known if N 2 fixation in this species is supported by the energy generated from H 2 oxidation. The presence of soluble hydrogenase which can generate N A D H for CO 2 fixation is an advantage to an organism growing under chemolithotrophic conditions. The absence of soluble NAD-dependent hydrogenase in P. saccharophila [179] suggests that the organism, when grown under chemolithotrophic conditions, has to gen-

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erate N A D H by proton motive force-dependent reverse electron flow [180]. Its chemolithotrophic N2-fixing growth was stimulated by the addition of nickel to a nickel-deficient culture medium [159]. Although chemolithotrophic H2-oxidising diazotrophs have been considered agriculturally unimportant [70], they could be significant in terms of contribution to the nitrogen economy and conservation of energy in aerobic or microaerobic ecosystems where organic carbon is limiting, CO 2 and H 2 supply is abundant [191], and nickel is sufficient. The rhizosphere could be one of these ecosystems. Watanabe et al. [149] reported the predominance of H2-utilizing bacteria among N2-fixing bacteria in wetland rice roots. He-oxidizing N2-fixing bacteria could be obtained in the rhizosphere of white beans [36] by enrichment isolation.

Denitrification Organic nitrogen in the biosphere is recycled via atmospheric N~ and nitrogen oxides by mineralisation, nitrification and denitrification. The latter process is usually carried out under O~-limiting or anaerobic conditions by facultative anaerobes which dissimilatorily reduce nitrate or nitrite in sequential steps to gaseous forms of nitrogen, mainly N2O and N 2. In this special form of anaerobic respiration, the nitrogen oxides are used as terminal electron acceptors instead of O 2 in oxidative metabolism coupled to phosphorylation. The most common and active soil denitritiers are pseudomonads, especially P. fluorescens biotype II, probably due to their great versatility in aerobic carbon metabolism rather than their anaerobic competence [40]. Hence, the carbon substrates for denitrification cover a wide range of natural products and synthesised chemicals [40,192]. Three of the N2-fixing pseudomonads listed in Table 2 (Pseudomonas sp. DC, 'P. diazotrophicus' H8 and P. stutzeri JM300) are known to be capable of denitrification. One of them, 'P. diazotrophicus' H8, has also been shown to denitrify chemolithotrophically [193]. The Pseudornonas

genus is not the only one that contains diazotrophs with the ability to denitrify [194]. The other diazotrophic genera containing denitrifiers are Aquaspirillum, Azospirillum, Bradyrhizobi,~m, Rhizobium (including R. fredii [195] which was proposed to be renamed Sinorhizobium fredii [196]), Rhodobacter and Rhodopseudornonas. (Although denitrifying strains are known to exist in the genus Agrobacterium [40] and results from one laboratory showed that several strains of A. tumefaciens can fix N 2 [103,104], independent verification of diazotrophy and confirmation of denitrification capability in each of these strains are required.) The relative contribution of these bacteria to the sources and sinks of soil nitrogen is difficult to assess. Their N:-fixing activity is mainly limited by energy source while denitrification is limited mostly by availability of reductant and nitrate, and presence of too much 0 2. It should be noted that aerobic denitrification can occur in t3~eudomonas aeruginosa [197] and Thiosphaera pantotropha (e.g. [198,199]). Simultaneous N~ fixation and denitrification in the same organism is probably rare. Although both activities have been measured in intact nodulated soybean plants [200], the two processes may have been carried out separately by differentiated or undifferentiated rhizobia, which are, respectively, bacteroids and free-living bacteria in the root nodules. Simultaneous N: fixation and denitrification by the same organism demands common physiological conditions that allow both processes to proceed, viz. (a) an O , concentration which is non-inhibitory to both processes; (b) an available nitrate concentration which is non-inhibitory to N 2 fixation; and (c) adequate electron flow to N~ and denitrification substrates. Since the diazotrophic pseudomonads are probably a minor componenl of the denitrifying population in soil, their contribution to total soil denitrification is expected to be small. The biological advantages of denitrification to N 2 fixers are not clear. Its value for pseudomonads in aiding saprophytic competence and surviving anaerobic conditions is questionable becausc these bacteria owe their competitiveness to their versatility in aerobic carbon metabolism rather than their ability to denitrify under specific and

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transient anaerobic conditions [40]. Rhizobial denitrification has been hypothesised to be potentially advantageous for maintaining nodule integrity under adverse 0 2 deficiency, alleviating nitrate inhibition of nodulation and nitrogenase, and supporting nitrogenase by ATP generation from coupled oxidative phosphorylation [201]. If proven, denitrification in rhizobia can be considered ancillary to symbiotic N 2 fixation. The recycling of N 2 released from denitrification through N 2 fixation has been demonstrated in Rhodobacter sphaeroides f. sp. denitrificans, indicating a localised nitrogen limitation [202]. Genes involved in denitrification, H 2 oxidation and CO 2 fixation are suggested to be mobile because of their location on a 450-kb plasmid in a non-diazotroph, Alcaligenes eutrophus H16 [203]. However, in the well-studied denitrifier P. stutzeri Zobell all the denitrification genes, except those encoding nitrate reductase function, are clustered and chromosomally located [204]. Recently, a DNA sequence homologous to that of the N 2 0 reductase structural gene from P. stutzeri was located on the nod megaplasmid, pSym-a, carrying genes essential for symbiotic N 2 fixation, in at least two well-characterised R. meliloti strains [205]. The similar general location of the denitrification and N 2 fixation genes in these rhizobial strains suggests that the two antagonistic nitrogen transformations may be under analogous genetic regulation. This is not totally unexpected since these two metabolic pathways share some common physiological requirements as stated above. Evidence is emerging for the involvement of global regulators, e.g. FNR and 0-54, in controlling the expression of the pseudomonad denitrification system in response to anaerobiosis and nitrogen oxides [204]. It will be interesting to know if similar controls also exist in denitrifying diazotrophic pseudomonds.

Conclusions

The existence of diazotrophs in the genus Pseudomonas is largely evidenced by the identification of putative N2-fixing strains of P. fluorescens, P. putida and P. stutzeri, species that

belong to the authentic Pseudomonas genus. Supportive evidence is also provided by N2-fixing strains genetically constructed from authentic members of the former two species, albeit they have low activity. However, definitive genus identification and nif gene expression remain critical criteria for the common recognition of diazotrophs in Pseudomonas species. A standard protocol for the rapid and reliable phenotyping and genotyping of an isolate is yet to be developed to facilitate its correct assignment in the genus Pseudomonas. It will also help to study the population dynamics of this ubiquitous group of bacteria. Presently, biochemical and genetic data on the pseudomonad nitrogenase are virtually non-existent. It is essential to characterise the enzyme and to analyse genetic determinants involved in diazotrophy by Pseudomonas species compared to those of known nitrogenase systems. Information on the regulatory genes of N 2 fixation, especially those which respond to 02, and the maintenance of all genetic determinants involved in nitrogenase function is also needed not only for assessing the significance of N 2 fixation by the Pseudomonas species, but also for understanding the evolution and distribution of diazotrophs. The contribution of N2-fixing pseudomonads to the nitrogen economy in specific ecosystems and in the long term has not been systematically assessed. Isogenic mutants and in situ 15N methodology would be indispensable for such investigations. Similarly, the relative contribution of diazotrophic pseudomonads which can also denitrify to the sources and sinks of atmospheric N 2 requires further clarification. Understanding of the possible coordinated genetic regulation of N 2 fixation and denitrification in the same organism may offer insight to modify the agronomically important rhizobia for enhancing N 2 fixation while reducing nitrogen loss through denitrification. It is believed that genetic studies of diazotrophic Pseudomonas species would shed new light on the control and improvement of N 2 fixation through the exploitation of their useful properties such as metabolic versatility and psychrotrophy. For example, increasing H 2 oxidation

110 as a n a n c i l l a r y f u n c t i o n t o N 2 f i x a t i o n b y g e n e t i c manipulation may augment N 2 fixation efficiency.

Acknowledgements The assistance of M. Graham in l i t e r a t u r e s e a r c h is v e r y m u c h a p p r e c i a t e d . T h i s a r t i c l e is c o n t r i b u t i o n N o . 1497 o f t h e P l a n t R e s e a r c h C e n tre, Agriculture Canada.

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