Impairment of transposon-induced mutants of Rhizobium leguminosarum

Soil Bid. Biockm. Vol. 23. No. 9. pp. 861-867, 1991 Printed in Great Britain. All rights resew4

IMPAIRMENT F. J.

a33&0717/91 53.00 + 0.00 Copyr$ht c 1991 Pcrgamon Press pk

OF TRANSPOSON-INDUCED MUTANTS OF RHIZOBIUM LEGCJMINOSARUM

BROCKMAN,‘*

L. B. FORSE.’ D. F. BEZDICEK’

and J. K. FREDRICKSON*

‘Department of Agronomy and Soils, Washington State University. Pullman. WA 99164 and 2Terrcstrial Sciences Section. K4-06. Pacific Northwest Laboratory. Richland, WA 99352. U.S.A. (Accepred 25 February

1991)

Summary-Tn5 mutagenesis was investigated as a technique to genetically mark Rhizabium for soil microcosm studies. Sixteen mutants created by Tn5 insertion or suicide vector integration were analyxcd to determine how the mutations affected several phcnotypic traits. These included growth rate in culture, symbiotic effectiveness.and competitiveness for nodule occupancy. Seven of IOTnJ-containing strains and 5 of 6 vector-integrate strains were impaired in one or more phenotypic traits in comparison to their parent. These results illustrate the need to carefully characterize genetically marked organisms instead of assuming the marked organism is simply an antibiotic-resistant strain qual to the parent.

INTRODUCTION

Transposons have been used in laboratory (Trevors. 1988; van Elsas er al., 1988; Bentjen er al.. 1989; Fredrickson er al., 1989; Pillai and Pepper, 1990) and field (van Elsas ef al.. 1986) studies to assess the environmental fate and effects of bacteria. Transposon-marked bacteria are easy to construct, have a low rate of reversion. are resistant to one or more antibiotics, and can be enumcratcd using DNA hybridization methods. However, it is not known how often random transposon insertions result in an organism that is less (or more) able to survive and compete in the soil environment relative to the parent. Fredrickson ef al. (1988) rcportcd a most-probablcnumber (MPN)-DNA hybridization technique that enumerated Tn5 mutant bacteria in soil at populations as low as IO-100 cells g soil-‘. Vectorintegrate strains (containing the transposon and the transposon delivery plasmid) were utilized because they enabled greater sensitivity in the MPN-DNA hybridization technique. We have determined the appropriateness of using transposon, and transposon vector-integrate, mutant bacteria for ecological studies of bacteria by initiating investigations of phenotypic changes (other than antibiotic resistance) in genetically-marked bacteria derived from parent strains. Genetically-marked derivative strains were constructed from the wildtype and from spontaneous antibiotic resistance (SAR) derivatives of the wildtype. SAR strains were included because detection of genetically-marked Rhizobium in soil using the MPNDNA hybridization technique may be improved if an additional selective marker was present (Fredrickson ef (II., 1988). Rhizobium spp offer an excellent system to test for transposon-induced phenotypic changes because a substantial portion of the bacterial genome encodes for functions in the legume symbiosis, and the efficiency of the bacterial-plant symbiosis can be *Present addras and author for correspondence: P.O. Box 999. K4-06. Pacific Northwest Laboratory. WA 99352. U.S.A.

Richland.

861

readily assayed. The objective of this study was to genetically mark Rhi:obium by Tn5 insertion or suicide vector integration and to determine how the mutations affected phenotypic traits, including growth rate in culture, symbiotic effectiveness. and competitiveness for nodule occupancy. MATERIAIS

AND MEWODS

Organisms, plosmids. matings and cultural

candirions

Bacterial strains and plasmids used and their relevant characteristics, arc presented in Table I. The transposon donor strain, Escherichia co/i MVIZ. containing the suicide vector pGS9. was obtained from M. Kahn (Washington State University, Pullman). The four Rhizobium recipient strains were obtained from D. Bczdicck (Washington State University. Pullman). Plasmids pCUlOl and pRZlO2 were also obtained from M. Kahn. Plasmids pCUl and pACYCl84 were obtained. respectively. from K. Bertrand (Washington State University. Pullman) and 8. lyer (Carleton University. Ottawa, Canada). Rhizobium strains were cultured on yeast extract mannitol (YEM) broth (Vincent, 1970) on a reciprocating shaker (90 rev min-‘) at 27°C. E. cali MV12 (pGS9) was cultured on Luria-Bcrtani (LB) broth (Miller, 1972) at 37°C. Antibiotic concentrations were 25 pg ml-’ for chloramphcnicol (Cm) and rifampicin (Rm). 5Opg ml-’ for kanamycin sulfate (Km) and 100 cg ml-’ for streptomycin sulfate (Sm) on liquid media, and twice that on solid media. Transposon mutagenesis was carried out as described by Fredrickson et al. (1988). Briefly, log-phase donor and recipient cells were washed, mixed and placed on sterile 0.45 pm pore-size nitrocellulosc filters on LB agar plates for 24 h at 27°C. Selection of transconjugants was made on Rhizobium minimal salts (RMS) agar medium (Sommerville and Kahn, 1983) supplemented with 0.5 g NH&I I-’ and 50 mg Km I-‘. Selection of DNA -marked derivative strains

Two classes of Km-resistant transconjugates were defined: those containing only Tn5 and those containing Tn5 plus vector DNA. The two classes were distinguished using three DNA probes: pGS9. pCUlOl and the 3.3 kilobase (kb) HindIiI internal fragm&tt of Tn5 (Jorgensen et al., 1979) (Fig. I). Km-resistant colonies were hybridized as described by Fredrickson et al. (1988). Twenty-five isolates (hereafter termed strains) from each class were

862

F. J. BROCKMAN cr al. Table I. Bacterial straim and plasmids wed

SVain E. R. R. R. R.

coli MVIZ(pCS9) kgwninosarum Ieguminosarwn lrguminosanm Ieguminos-

biovar biovar biovar biovar

viceat IP vicroc IP-RS phawoli Kim-5 phmeoli Kim-5-R

Relevant markcn/dwiation

R&retNX

Icu-thr-thi-crpthywildtype Rm’ Sm’ derivative of I P’ wildtype Rm’ derivative of Kim-5

Sommcrville and Kahn (1983) this lab. (DFB) Turco lr al. (1986) this lab. (DFB) this lab. (DFB)

Cm’ Km’ pClJlOl::TnS see Fig. I see Fig. I see Fig. I see Fig. I

Selvaraj and lycr (1983) Konanka-Kozlowska and lyer (1981) Konarska-Kozlowska and lycr (1981) Chang and Cohen (1978) Jorgensen CI al. (1979)

Plasmid pGS9 pCtJlOl

PcUl pACYCI84 pRZlO?

‘Abbreviations: Rm. rifampicin;Sm. streptomycin sulfate; Cm. chloramphenicol; Km. kanamycin sulfate; upper ease r. resistant.

individually grown for approx. 150 generations by successive transfers on non-selective RMS broth and then grown on RMS agar plates. For each strain, 96 colonies were replica picked to YEM. YEM-Km, and when appropriate, YEM-Km-Cm to test for maintenance of the antibioticresistance markers. From the 25 strains from each class, 10 TnS-only strains and 6 transposon vector-integrate strains showing no marker loss (i.e. reversion frequency sensitivity of IO-*) were randomly selected for further study.

Growth rate in culture was determined by inoculating 10 ml of unamendcd YEM in a SO-ml Erlenmeyer flask with 10~1 of log-phase culture. Flasks were kept at 27°C and shaken at 90 rev min-‘. Growth curves were constructed from dilution plate counts in triplicate every 2 h. Linear regression of cell growth during log phase was uxd to calculate

doubling

time of the strains.

The symbiotic elTectivcness of individual strains was studied in sterile. modified Leonard jars containing vermiculitc, sand (stcrilc. I: I v/v) and sterile N-free nutrient solution (Leonard. 1943; Slogcr. 1969). Pisum sufiuum cv. Alaska and Phuseolus udguris cv. Viva Pink seeds were surface disinfected in 0.5% hypochlorite for 10 min. washed 5 times with sterile water, and placed between wetted sterile toweling in Petri dishes. This method had proved to be effective in surface disinfecting >95% of the seeds prior to germination. After 3 days, 2 seeds with radicles emerged were planted aseptically 2.5 cm deep in each of three replicate Leonard jars. Log-phase cultures were washed in sterile water, counted by hemacytomcter. and 2 x 10’ cells were applied in 500~1 YEM broth to each seed. Plants were grown without supplemental lighting during July and August in a greenhouse maintained at 30 and 18°C (&3”) during the day and night, respectively. To minimize edge effects, the jars were rotated to edge, middle, and central positions weekly. Plants were harvested at mid-pod fill. 38 and 50 days after planting, respectively, for pea and bean.

HH I to I pGS9

1

pCUlO1

-

3.3

HEH I 1;“’ 2.5 I

Nodule number, plant dry weight, percent tissue N, C:N ratio, and total tissue N were determined. N and C contents were determined with a Leco CHN-600 Combustion Autoanalyzer (Leco Corporation, St Joseph, Mich.). The maintenance of the Km-resistant and Cm-resistant markers was assessed in this experiment to determine whether in-soil or in-planta loss of marker DNA could occur, and if putative wildtype isolates recovered from nodules in the nodulation experiment (see following section) could be due to excision of marker DNA. At plant harvest, 16 nodules were removed from each of 6 plants in each treatment. Nodules at root apices farthest from the soil surface were selected to represent isolates with maximum residence time in the soil-rhizosphere before nodulation. Each nodule was surface-disinfected in 2.6% hypochloritc for I min. washed 5 times with sterile water. and transferred to 100 ~1 of saline in the well of a microtitre tray. Surface disinfection by this method had proved to be effective for disinfection of > 95% of the nodules. Nodules were crushed, and the resultant suspensions were replica plated onto YEM. YEM-Km. and when appropriate, YEM-Km-Cm. Confluent growth on YEM and YEM containing antibiotics indicated that the bacterium which colonized the nodule had rctaincd the DNA markers. Bacteria unable to cxprcss Km resistanceor Cm resistanceon YEM containing antibiotic(s) were analyzed for loss of marker DNA by hybridization analysis. Nodule occupancy

Competition for nodule occupancy was studied in Leonard jars as described in the symbiotic effectivenesssection. except for the following modifications. Each marked strain (10 genetically-marked and 2 SAR-marked) was individually challenged with the wildtype by co-inoculating a I:1 mixture (2 x IO’ cells of marked strain and wildtype) on each seed. At plant harvest, nodules from the upper tap root were selected to represent the early rhizosphere composition. To assess whether nodule double occupancy was contributing to the putative recovery of marked isolates, an

H

pcu I pACYC184

-

pRZ102 Fig. I. Linear&d pGS9 showing location of restriction enzyme cleavage sites, size of restriction fragments in kilobases, and sequences corresponding to plasmids used as hybridization probes. Large open box = TnS, short open box = truncated copy of ISSO; H = HindHI; E = EcoRl.

Rhizobium&guminosarwn

Transposon-induced

additional experiment was conducted. Each SAR parent was individually challenged with its genetically-marked derivative strains by co-inoculating a I : I mixture (2 x IO’ cells of SAR strain and SAR genetically-marked strain) on each of two seeds in a single Leonard jar. At plant harvest. 48 nodules were removed from each of two plants in each treatment. and the nodules handled and resultant bacteria charactcrixcd as described. Bacteria were replica plated to YEM-Rrn (for SAR parent) and YEM-Km. Growth on both scelcctive media indicated dobule occupancy. Growth rate, symbiotic effectiveness. and nodule occupancy experiments were initiated within 36 h of each other from YEM cultures kept at 4-C after attainment of log phase growth. Following experimental setup, cultures were streaked to YEM to verify culture purity. Generic

RE!WLrS

time. maintcnancc and

vector

of gcncfic markers.

inlcgration

(VI)

strains

and nodule

on pea and

Strain

lime

wildlypc parent VI VI Tn5 Tn5 SAR

IP-I I P-2 IP-J I P-4

IP.RS IP-RSI

VI Tn5 TnJ Tn5

I P-RS? IP-RSJ I P-R!34

wildlype parent VI TnJ Tn5 Tn5 SAR

Kim-S Kim-S-l Kim-s-2 Kim-5-3 Kim-S-4 Kim-S-R

Kim-S-R2 Kim-S-R) Kim-S-R4

(min)’

Tn.tb

loss

Nod&

pculol~

occupancyd

9x

NA’

NA

NA

157’

0

8

4R

96

0

6

51

144’

0

NA

57

86

0

NA

41

97

NA

NA

4”

83

ND’

ND’

W”

153’

0

NA

3’

I04

0

NA

I’

146’

0

NA

(r

86

NA 0 0 0 0 NA

NA

NA

2

46

90 100 87

a3 153’

VI Vi Tn5 TnJ

Kim-S-RI

ofTnS-only

Dnuhling

wnolync

IP

occupancy

bean planlr

Marker Rclcvant

of sfruim

Doubling time was delayed significantly (P = 0.05) in 5 of the 16 genetically marked strains (IP-I, 1P-3. I P-R2. 1P-R4, Kim-5-R3) and in Kim-S-R (Table 2). Significant changes in one or more symbiotic effectiveness characteristics were observed in 5 of 6 vectorintegrate (VI) strains and in 2 of 10 TnS-only strains in comparison to their immediate parent (Table 3). Strain lP-RSI was phenotypically nod- fix-, while strains Kim-5-RI and Kim-5-R2 were impaired in nodulation but fix-. In addition to these strains, significantly different values were observed in four strains (IP-I. IP-RS, Kim-S-l, Kim-5-R4) for plant dry weight, three strains (Kim-5-l. Kim-5-2, Kim-SR) for tissue N. two strains (Kim-S-l. Kim-5-R) for C:N ratio, and four strains (IP-I, Kim-53, Kim-5-R. Kim-5-R4) for total N. Strain Kim-5-l exhibited increased plant dry weight but decreased tissue N. and thus total N was not statistically different. Strain Kim-5-2 exhibited increased tissue N and increased total N. Four of the five genetically-marked strains with dccreascd growth rate were not impaired in any symbiotic effectiveness property, indicating that growth rate in culture was a poor predictor of impairment in symbiotic eflcctiveness. Statistically significant differences in values for total plant N were

characrertarion

2. Doubling

chafucferixfion

Biochemical

Plasmid visualization using a modification (Fredrickson er al., 1988) of Eckhart in-well lysis (Eckhart. 1978) and Southern hybridization analysis with pGS9 as a probe were used to determine the replicon that contained marker DNA. Genomic DNA was prepared by the method of Currier and Nester (1976). restricted with Hindlll or EcoRI. and analyzed by Southern hybridization with pGS9. pCUlO1. pCUl. pACYCl84 and pRZl02 to determine if each strain represented a different insertion loci. if duplicated regions of pGS9 were present. and if pGS9 sequences were present at multiple insertion loci. The relationships bctwcen plasmids used as hybridization probes arc depicted in Fig. I. Plasmid preparation. ‘*P-labeling of probes. dot blots and Southern blots. and hybridization were performed as described by Fredrickson et (11. (1988).

Tahlc

863

NA

28”

NA

31”

NA

s2

NA

42

0

I32 II3

9” 27”

214’

N’ A

44

142

NA

52

‘Minutes required for doubling from linear regression oflog phase growth. The coeflicicnt of I3 (i) was typically 0.98 or greater. allowing comparisons between strains IO be made with the chi square ICSI. bNumber of nodules (IOIA - 96) colonized by a bacterium that had 10~1 TnS. ‘Number of nodules (lo131= 96) colon&d by a bacterium that had lost pCUlOl sequencer. ‘Number of nodules (total - 96) occunied bv DNA-marked strain. ‘Not applicable. ’ ’ ‘Impairment in growth was inferred when there was a statistically significant difference (P - 0.05 by x2 test) between the immediate parent (i.e. IP. IP-RS. Kim-J, Kim-S-R) and the DNA-marked ‘Strain

was nod-

‘Biochemical

impairment

(P - 0.05 Kim-S. ‘The

by x1 tat) Kim-S-R)

very low for

derivative (see Table

nodule

nodule

strain.

3).

was inferred between

nodule

when

and the DNA-marked occupancy

aruponcy

by

there

occupancy

I P-RS

for these strains.

was a statistically by the immediate

derivative precluded

significant parent

diticrence

(i.e. IP.

IP-RS,

strain. statistical

analysis

of competitiveness

F. J. BROCKMAS et d

864

Tabk 3. Symbiotic c&ctivcncss of TnS-only and vector integration (VI) strains on pea and bean plants’ Noduk number (plant - ’ )

Strain IP IP-I IP-2 I P-3 I P-4 IP-RS’

wildtyp VI VI Tn5 TnJ SAR

IP-RSI I P-RS2 I P-RS3 IP-RS4

VI Tn5 TnJ Tn5

Dry weight (g plant-’ )

Tissue N

(%I

C:N ratio

Total N (mnJ

254 240 249 252 249 251 Ob.’

7.8 6.t-F 8.1 8.7 8.0 6.6d

3.1 3.2 3.0 3.1 3.1 3.3

13.5 12.9 14.0 13.4 13.2 13.3

238 IW 240 269 250 20s

I .Ob,’

261 244 212

6.4 6.7 6.4

I .4’ 2.9 3.9 2.9

26.4b 14.0 14.4 14.2

I 5b” I88 193 188

I.3

3.6

10.8

48

Kim-S Kim-5-I Kim-S-2 Kim-S-3 Kim-54 Kim-S-R

wildtype VI Tn5 Tn5 Tn5 SAR

413 420 377 407 393 423

6.8 8.01 6.9 7.0 6.3 6.1

4.6 3.Sb S.IC 4.8 4.9 4.0b

a.1 l0.7b

311 278 3536 336 305 248’

Kim-J-RI Kim-S-R2 Kim-S-R3 Kim-S-R4

VI VI Tn5 Tn5

l73b 317c 400 417

1.7b’ 3.sJ 6.5 5.2d

1.9bS 2.ab 4.1 3.9

l9.7b l3.3b 9.6 9.9

I.3

I.5

22.9

Uninoculated control

Uninoculated control

0

0

7.8 8.5 a.4 9.8d

33b” 98b 264 2OOd 23

‘ANOVA contrast test: “‘,“dcnotc a significant difkrencc from immediate parent (i.e. IP, IP-RS. Kim-s. Kim-J-R) at P -0.01. 0.05 and 0.10. respectively. Values statisticnlly similar to the uninoculated control arc denoted by *. Each value is for a total of 6 plnnts contained in triplicate Leonard jars of 2 plants jar-‘. ‘Strain IP-RS was previously selected for arowth rate and symbiotic etlectivencsssimllw IO the wildtypc parent (Turco CI ul.. 1986;

often not rcflectcd in nodule number (i.c. strains I P-l, Kim-S-2, Kim-5-R. Kim-S-R4). and dccrcascs in the number of nodules. although significant, wcrc not reflective of the dcgrcc of dccreascd total N in strains Kim-5-RI and Kim-S-R2. These observations indicate that nodule number did not always predict impairment in symbiotic effectiveness. Nodule mass was not recorded because the distribution of nodule sizes produced by all fix+ strains appeared to be closely similar. Competition for nodule occupancy was reduced in 3 of 6 VI strains, 2 of 7 TnS-only strains, and in strain IP-RS (Table 2). Both nod+ fix- strains (Kim-S-RI

and Kim-S-R2) exhibited decreased competitiveness. Strains IP-RS, Kim-5-2, and Kim-5-3 exhibited reduced competitiveness, yet these strains did not produce fewer nodules or fix less N (Table 3). Therefore, competition for nodule occupancy defined biochemically-impaired strains which the growth rate and symbiotic effectiveness properties did not. The frequency of nodule double occupancy was sufficiently low, o-4% in all but one treatment (data not shown), to not affect statistical analysis. Genetically-marked bacteria from nodules farthest from the soil surface maintained Km-resistance (Km’) in all but one of 1440 nodules examined, and maintained Cm’ in all but 17 of 480 nodules examined (Table 2). Hybridization analysis showed that bacteria present in these I7 nodules had lost pCUlOl sequences, yet Tn5 had been retained in all but one instance (data not shown). Thus, although loss of the Cm’ marker was significant, marker loss by genetically-marked bacteria did not significantly contribute to the total number of presumptive wildtype isolates from nodules.

When results of the growth rate. symbiotic effcctivcncss, and competition cxpcrimcnts wcrc assessed

collcctivcly. 5 of 6 VI strains and 7 of IO TnS-only strains wcrc impaired rclativc to their parent. Although the sample sizes wcrc rclativcly small, VI strains displayed more frequent and more severe impairment in symbiotic cffcctivcness and compe tition for nodule occupancy than did TnS-only strains. Genetic churacteri:ation

of strains

Approximately 4% of the Km’ isolates produced a more intense autoradiographic signal than the other Km’ isolates after hybridization with the pGS9 probe, yet all Km’ isolates produced signals of equal intensity when probed with the Hind111 fragment of Tn5 (data not shown). Only those isolates displaying intense hybridization with the pGS9 probe hybridized with pCUIO1. indicating the maintenance of vector sequences in these isolates. Southern hybridization analysis of Eckhart-type gels with the pGS9 probe demonstrated that introduced DNA was chromosomally located, and that vector sequences had not been maintained as an autonomous plasmid (data not shown). Because multiple insertion loci could account for the more frequent and more severe impairment in VI strains. the relative location of inserted DNA in the chromosome was further examined. Hybridization analysis of HindIII-digested genomic DNA with the pGS9 probe indicated that all genetically-marked strains were unique, with the exception of strains IP-RS2 and lP-4. which appeared to share identical insertion loci (Fig. 2). A single copy of an intact Tn5 was present in each of these strains. The linearized

Transposon-induced Rhtiobium Ieguminosurum

865

Fig. 2. HinJlll genomic digests hybridized with pGS9. L;tne I IP-I; lane 2 IP-RSZ: lane 3 IP-RSI; lane 4 IP-4; lane 5 IP-RS.1: lane 6 IP-KM; lane 7 Kim-S-RI; lune 8 Kim-S-R?: lane 9 Kim-S-2 lane IO Kim-S-J: lane I I Kim-5-R4; lane I2 Ilindlll-dig&cd pGS9. At left are the Ili~rrllll fragments of phage lambda and their sizes in kilobascs.

vector (i.e. pGS9 fragments of 20.5. 4.3, 3.3, and 2.0 kb; see Fig. I) was contiguous in VI strains IP-I. IP-RSI. Kim-5-RI, and (in an autoradiograph not shown) strains I P-2 and Kim-S- I. Thcsc strains also posscsscd a fifth fragment of variable size, indicating the presence of a duplicated scqucnce in addition to the contiguous linear&d vector. VI strain Kim-S-R2 contained five fragments of 16.1, II& 4.3. 3.3, and 2.0 kb (lightly hybridizing bands in Fig. 2 wcrc determined to be a result of incomplctc digestion). Hybridization analysis of HindIll-digested genomic DNA with pRZlO2 and pCUIOI as probes dcfincd the duplicated region as a portion of Tn5 in strains IP-I. IP-RSI. Kim-5-l. and Kim-5-RI; strain Kim-5R2 was found to contain duplicated regions corrcsponding to a portion of both TnSand pCU 101 (data not shown). To investigate whether the duplicated regions in VI strains were contiguous with the chromosomallyintegrated vector or present at a non-contiguous locus, Southern hybridization analysis of Eco Rl-digested genomic DNA was carried out with pRZIO2, pCUl and pACYCl84 as probes. Figure I shows that Eco RI cleavage of chromosomally integrated pGS9 would produce two fragments. Plasmic GUI would hybridize only to the fragment to the right of the EcoRI site; pACYCl84 would hybridize to both fragments, as would pRZlO2 (from hybridization to the copy of the IS50 element of Tn5) (Fig. I). If the duplicated region was contiguous with the chromosomally-integrated vector, or nearly so, such that an EcoRI site was not present in the intervening genomic DNA, an additional hybridization band would not be found. Hybridization analysis showed the expected number of bands with all three probes

(results with pRZIO2 shown in Fig. 3). which dcmonstratrd that the duplicated portion of Tn5 in strains IP-I, IP-RSI, Kim-5-l. and Kim-5-RI was contiguous (or nearly so) with the vector. Strain Kim-S-RI displayed an additional band with only the pRZlO2 probe (Fig. 3). which dcmonstratcd that the duplicated portion of TnS was at a non-contiguous locus and the duplicated portion of the pCU IO I region was contiguous (or nearly so) with the vector. tMSCUSSlON The validity of stud& that USC a transposon marker to track the environmental fate of bacteria r&s on the assumption that the marked bacteria and the wildtype arc equally competent under the existing environmental conditions. Our results indicate that strains derived by transposon mutagenesis can not be assumed to be antibiotic resistant strains that arc identical to the parent in growth rate. symbiotic effectiveness. and competitiveness for nodule occupancy. Thcsc results paralkl the finding that SAR mutants of Rhkobium were not identical to the parent in growth rate, symbiotic cffcctivenizss. and compctitivcncss (Turco et al.. 1986). In addition, Pseud~~n~onus Rm SAR mutants can exhibit altcrcd membrane protein composition. decreased growth rate. and decreased ability to compctc with wildtype parents in stcrilc soil assays (Compeau PI al.. 1988). In our study with Rhizobium transposon mutants, each of the three measurements growth rate, symbiotic effectiveness, and competitiveness identified impaired strains that the other two measurements did not, emphasizing the need for multiple measures to assay competence. Although growth rate in culture is

F.J. BROCKHM

866

et al.

23.1 -

6.7 -

4.3 -

2.2 -

2.0

Fig. 3. EcoKf

lane 4 II’-I:

-

gcnomic digests hybridized with pRZIO1. Lsnc I Kim-S-RI; lane 2 Kim-5-l; lane 3 IP-RSI; lane 5 Kim-S-RZ. At left arc the NinJlII f’ragmcnts ol’ phagc lambda and their siza in kilohuscs.

probably a poor predictor of compctcncc in the environment. it was included in this study bccausc many studies rely solcly on growth rate to conclude that a transporon-containing strain is cquivalcnt to the parent. The high frcqucncy of impaired transposon mutants obscrvcd in our study may result from the ability to cl?icicntly assay for functions rcprcscnting a relatively large portion of the Rhimbium genomc. Similar analysts would bc difficult to conduct for many other soil bacteria which lack a suite of functions (other than growth rate) that can be readily assayed. For thcsc bacteria. more sophisticated methods must bc developed to determine if the transposon mutant and parent are of equal compctcnce. Furthcrmorc. methods should address the fact that impairment may bc dithcuh or impossible to dctcrminc in stcrilc or gnotobiotic conditions due to lack of competition with indigenous organisms etc., and may lx mnnifcstcd only in the natural environment. Thcsc concerns also apply to gcnctically cngineercd bacteria, as the introduction of foreign gcncs often relics on either a random transposon target site (Watrud er cf., 1985; Barry, 1986) or a restriction enzyme site (Drahos er al.. 1986; Holbcn er al., 1988) in DNA that codes for an unknown function. One approach to characterize transposon-marked (or gcnctically-cnginccrcd) bacteria would bc to con-

duct an initial (non-stcrilc) microcosm study to verify the assumption that the sclcctcd strain is equivalent to the parent (e.g. Pillai and Pcppcr, 1990). A second approach would bc to forego the initial study and co-inoculate with several diffcrcnt transposonmarked (or genetically-engineered) strains to increase the likelihood of using environmentally-competent strains (Bcntjcn et (II., 1989; Fredrickson et al.. 1989). Although chromosomal integration of pGS9 has been reported by Cuppcls (1986). Rostas ef al. (1984) and Sclvaraj and Iycr (1983). we were the first to cxaminc the etTccts of vector integration on biochemical impairment. Bccausc the sample size was small. only qualitative statements can bc made. NcverthcIcss. TnS-containing strains tended to bc more like the parent and more likely to maintain marker DNA than VI strains. The greater frcqucncy and severity of impairment in VI strains may bc a result of increased polarity. as only strain Kim-S-R? exhibited multiple insertion loci. Howcvcr, it is possible that the other VI strains had multiple. but closely linked, insertion loci that our analysis was not sensitive enough to detect. For thcsc reasons. the use of VI strains in ecological studies should bc qurstioncd. and hybridization probes should bc sclcctcd to allow for the diIfcrentiation of transposon-containing strains and VI strains.

Transposon-induced

Rhtiobiurn

AcknowledgemenlJ-We thank Dr David Herron for his valuable review of this manuscript, and Craig Root for assistance in statistical analysis. This research was supported

by a U.S. Agency for International Development/U.S. Department of Agriculture/Crop Sciences Research Society joint project on biological nitrogen fixation and the U.S. Department

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