Inactivation of the Regulatory Gene algU or gacA Can Affect the Ability of Biocontrol Pseudomonas fluorescens CHA0 To Persist as Culturable Cells in Nonsterile Soil

Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutantsin which the regulatory gene algU (encoding sigma factor ${sigma}$ ^E)or gacA (encoding a global regulator of secondary metabolism)was inactivated were compared for persistence in three nonsterilesoils. Functional algU and (particularly) gacA were needed forCHA0-Rif to maintain cell culturability in soil.

INTRODUCTION

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Introduction

Certain fluorescent pseudomonads are important for bioremediationof polluted soils or biological control of soilborne phytopathogenicfungi (5, 11). Once in the soil, pseudomonads often undergoa reduction in CFU, and in some cases this is due to the lossof the ability of cells to form a colony on laboratory mediaroutinely used to grow the inoculant (1, 7, 20). Indeed, a significantproportion of the nonculturable cells may respond positivelyto a viability test, suggesting that they are not dead (1, 5,20). Cell viability is often assessed by using Kogure's cellelongation test (10), which is based on the assumption thatviable cells can respond to nutrient stimulation and engagein a few cell divisions, without necessarily exhibiting colony-formingability. In this test, nutrient-responsive cells (consideredas viable) are prevented from dividing by the presence of nalidixicacid, enlarge significantly, and can be distinguished from smaller,nonresponsive cells. In this context, the occurrence of cellsin a viable but nonculturable (VBNC) state, as defined by Oliver(17), can be derived from the difference between viable countsand CFU of the strain.

The significance of VBNC cells remains to be elucidated (13).It has been hypothesized that the VBNC state(s) could representan adaptative strategy under adverse conditions. For the biocontrolagent Pseudomonas fluorescens CHA0, this possibility was suggestedby the fact that, in the field, the number of VBNC cells exceededCFU by several orders of magnitude for more than 6 months (20).The hypothesis is in accordance with observations that (i) deadcells introduced into nonsterile soil disappear rapidly (21)and (ii) VBNC cells may recover their previous colony-formingability (17), although such "resuscitation" experiments areprone to experimental artifacts (3). Alternatively, other authorshave proposed that VBNC cells are dying cells (13).

In some cases, the loss of colony-forming ability is thoughtto be an experimental artifact caused by exposure to oxidativestress on the plates (2), especially for H₂O₂-sensitive culturablecells (3); the stress may be alleviated by adding antioxidantsto the plates (3). However, exposure of CHA0 to H₂O₂ does notlead to VBNC cells (7), and the addition of various antioxidantsand radical scavengers when samples containing putative VBNCcells are studied does not improve the recovery of CFU (unpublishedresults). Rather, field observations have suggested that theoccurrence of VBNC cells in this pseudomonad was caused by exposureto stress conditions in situ (5, 20), which was confirmed experimentallyin vitro (12) and in soil (unpublished results). The formationof VBNC cells seems induced when stress intensity is sufficientlyhigh (12), which implies that up to a particular threshold ofstress, regulatory systems manage to adapt cell physiology andcell culturability is maintained. If this were true, it couldmean that VBNC cells may occur even at a lower stress levelprovided that relevant regulatory systems are inactivated. Alternatively,their inactivation would cause cell death, as often assumed(8, 16). CHA0 is a soil inhabitant and thus is adapted to thestressful conditions prevailing in this habitat. Therefore,the objective was to assess whether mutational inactivationof key regulatory genes could affect the ability of CHA0 topersist as culturable cells in nonsterile soil.

We chose to focus on two regulatory genes, algU and gacA. ThealgU (algT) gene codes for the alternative sigma factor ${sigma}$ ^E ( ${sigma}$ ²²),which regulates transcription of the heat shock sigma factor ${sigma}$ ^H ( ${sigma}$ ³²) and extracytoplasmic stress response (8, 14). In pseudomonads,algU controls alginate production (8, 18). The global activatorgene gacA (encoding a response regulator) is required for thesynthesis of extracellular enzymes and antimicrobial secondarymetabolites in CHA0 (11). Mutational inactivation of gacA almostcompletely eliminates the ability of CHA0 to protect tobaccofrom black root rot (11), and in soil CFU of the gacA mutantCHA96-Rif decreased more rapidly than those of the gacA⁺ strainCHA0-Rif (16).

A spontaneous rifampin-resistant mutant of CHA0 (i.e., CHA0-Rif[16]) was used so that the inoculant could be recovered fromnonsterile soil. A gacA'-'lacZ translational fusion was introducedinto CHA0 by gene replacement, resulting in CHA96 (11). StrainCHA96-Rif, a spontaneous rifampin-resistant mutant of CHA96,has already been investigated in soil and in vitro grows likeCHA96 (16). The algU gene has been cloned in CHA0 (18). StrainCHA212-Rif is an in-frame deletion (250-bp) algU mutant of CHA0-Rifobtained by gene replacement (18). Both strains have the samegrowth rate in rich medium. Inocula consisted of mid-log-phasecells harvested from King's B agar (9) containing rifampin andwashed in sterile distilled water (12).

Soil was collected in November from three Swiss cambisols (Table1) located at Eschikon, Tänikon, and Mellstorf. At Mellstorf,the soil was taken immediately below the forest litter, andit differed from the two farm soils, particularly on the basisof pH and organic matter content and quality. Sterile distilledwater was added to sieved (5-mm pore size) soil (6, 12) to achievea water potential of -0.03 MPa (Table 1) after subsequent additionof inoculum, as described previously (6, 12). Each soil received3.3 ml of cell suspension of CHA0-Rif, CHA96-Rif, or CHA212-Rif(i.e., 10⁸ cells added per g of soil) or sterile distilled water(uninoculated control) per 100 g of soil. Each soil was thoroughlymixed and 10-g samples were distributed into 25-ml vials. Conditionsof incubation (12°C, relative humidity of 70%) of the vialswere as described previously (6, 12). During the 60-day experiment,soil water content remained essentially constant (±0.1%[wt/wt]).

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TABLE 1. Main characteristics of the three soils examined in this study^a

Sampling, cell extraction, and dilution series were done asdescribed previously (12). CFU were obtained on King's B agarcontaining rifampin (100 µg/ml) and the antifungal compoundcycloheximide (190 µg/ml) after the plates were incubatedat 27°C for 2 to 7 days. In the uninoculated control, norifampin-resistant colonies were found. The total number ofCHA0-Rif cells was determined by immunofluorescence microscopy(12, 20) with a primary antiserum specific for CHA0 (19). Nocross-reaction was found in uninoculated soil samples. The numberof viable cells of CHA0-Rif was determined by using a combinationof Kogure's viability test (10) and immunofluorescence microscopy,as described previously (6, 12). Most inoculant cells in soilwere <1.5 µm in length. Substrate-responsive cellselongated to $>=$ 3.5 µm, and they were counted as viable.When population levels were low, filters were examined untilat least 10 elongated cells were found.

All treatments were replicated three times (destructive sampling)along a randomized block design. Cell counts were expressedper gram of dry soil and log transformed. Three-way analysesof variance (strain by soil by cell enumeration method) weredone at each sampling, and Tukey's honestly significant differencetests were used (when appropriate) to compare treatments (P< 0.05; SYSTATS version 5, SPSS Science, Chicago, Ill.).First, data from each soil were analyzed at each sampling foreach cell count method. Data and the results of statisticalanalyses are shown only for CFU (Fig. 1). Second, the influenceof the cell count method was assessed for each strain by comparingtotal counts, viable counts, and CFU at each sampling in eachsoil. Data and the results of statistical analyses are shownonly for the last sampling (Fig. 2).

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FIG. 1. CFU of P. fluorescens CHA0-Rif, the algU mutant CHA212-Rif, and the gacA mutant CHA96-Rif for 60 days after inoculation in Eschikon soil (A), Tänikon soil (B), and Mellstorf soil (C). The detection limit was 10² CFU/g of soil. Error bars show standard deviations. Statistically significant differences between strains were found at 60 days and are indicated by the lowercase letters a, b, and c.

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FIG. 2. Persistence of P. fluorescens CHA0-Rif, the algU mutant CHA212-Rif, and the gacA mutant CHA96-Rif at 60 days after inoculation in Eschikon soil (A), Tänikon soil (B), and Mellstorf soil (C). The numbers of total cells (immunofluorescence microscopy), viable cells (Kogure's test), and CFU of the inoculants were determined. The error bars show the standard deviations. Data were analyzed separately for each soil. For each strain, statistically significant differences between cell counts (i.e., total counts, viable counts, and CFU) are indicated by the lowercase letters a, b, and c. For each type of cell count, statistically significant differences between the three strains are indicated by the Greek letters ${alpha}$ , ß, and ${gamma}$ .

Effect of algU inactivation.

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Effect of algu inactivation.

The algU mutant CHA212-Rif, like CHA0-Rif, persisted as culturablecells in Eschikon and Tänikon soils (Fig. 1 and 2). InMellstorf forest soil, CFU of CHA212-Rif decreased by about2 log units from 30 to 60 days, whereas those of CHA0-Rif remainedstable (Fig. 1C). On day 60, CHA0-Rif was still culturable buttotal counts and viable counts were higher than CFU for CHA212-Rif(Fig. 2C), indicating the presence of VBNC cells of CHA212-Rif.

Therefore, a functional algU gene was not required to maintaincell culturability in the two farm soils but was apparentlyneeded in Mellstorf forest soil, where VBNC cells were 10 timesmore numerous than culturable cells for CHA212-Rif. This suggeststhat the inoculant was exposed to stressful environmental conditionsin the forest soil, perhaps because of the low pH or organicmatter quality. Indeed, algU controls tolerance to differenttypes of environmental stress in pseudomonads (8, 18).

Effect of gacA inactivation.

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Effect of gaca inactivation.

CFU of the gacA mutant CHA96-Rif decreased with time in thethree soils, especially in Mellstorf soil (Fig. 1). In Eschikonsoil, VBNC cells were found at 60 days (but not at 30 days [datanot shown]), as total counts and viable counts were higher thanCFU by >1 log unit (Fig. 2A). The same trend was observedin Tänikon soil, except that the difference between viablecounts and CFU was not statistically significant on day 60 (Fig.2B). In Mellstorf soil, there was a 4-log unit difference betweenviable counts and CFU of CHA96-Rif at 60 days (Fig. 2C), andVBNC cells were already evident on days 15 and 30 (data notshown).

In contrast to CHA0-Rif and CHA212-Rif, the gacA mutant CHA96-Rifwas clearly compromised for survival in all three soils, sinceCFU were lower than those of CHA0-Rif. This result is reminiscentof data from stationary-phase competition experiments in vitro(4). In Eschikon and Mellstorf soils, viable counts of CHA96-Rifexceeded CFU, and VBNC cells could represent $>=$ 10⁶ cells/g ofsoil. A spontaneous gacS mutant persisted like the gacA mutantin the three soils (data not shown), in accordance with thefacts that (i) GacS is the cognate transmembrane sensor kinaseactivating GacA and (ii) mutations in either gene lead to thesame phenotype in CHA0 (4, 11). The possibility that gacA couldcontribute to stress resistance in CHA0 is raised by the observationthat, in P. fluorescens Pf-5, gacA regulates positively theaccumulation of the stationary-phase sigma factor ${sigma}$ ^S ( ${sigma}$ ³⁸) andcontributes to oxidative stress resistance (22). This may beparticularly relevant to explain the results obtained in Mellstorfforest soil.

Almost nothing is known of the genetic factors implicated inthe formation of VBNC cells (15). We show here that algU and,particularly, gacA contribute significantly to the capacityof CHA0-Rif to persist as culturable cells in soil, and it isproposed that the occurrence of VBNC cells of the algU and gacAmutants may result from their inability to adapt their cellphysiology in response to environmental stress.

ACKNOWLEDGMENTS

We thank Matthias Riesen and Mathias Gasser for technical assistance.

This work was supported by the Swiss Biotechnology PP (project5002-04502311), the Swiss Federal Office for Education and Science(project IMPACT 2 and COST Action 830), and the French Embassyin Switzerland (France-Switzerland research grant).

FOOTNOTES

* Corresponding author. Mailing address: Phytopathology Group, Institute of Plant Science, Swiss Federal Institute of Technology (ETH), Universitätstr. 2, LFW, CH-8092 Zürich, Switzerland. Phone: 41-1-632-38-69. Fax: 41-1-632-11-08. E-mail: genevieve.defago{at}ipw.agrl.ethz.ch.

Present address: Swiss Federal Research Station for Plant Productionof Changins (RAC), CH-1260 Nyon, Switzerland.

REFERENCES

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