Department of Plant Biology, Swedish University of Agricultural Sciences (SLU), PO Box 7080,SE-75007 Uppsala, Sweden 1
Institute of Molecular and Cell Biology, University of Tartu, 23 Riia Street, EE 2400 Tartu, Republic of Estonia2
Author for correspondence: Minna Pirhonen. Tel: +46 18 673316. Fax: +46 18 673279. e-mail: Minna.Pirhonen{at}vbiol.slu.se
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ABSTRACT |
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Keywords: extracellular enzymes, pathogenicity, sigma factor, catalase, competition
Abbreviations: Amp, ampicillin; Cel, cellulase; Cm, chloramphenicol ; EAI, Erwinia autoinducer; Km, kanamycin ; Peh, polygalacturonase; Pel, pectate lyase; PGA, polygalacturonic acid; Pnl, pectin lyase; Prt, protease; SA, salicylic acid
The GenBank accession number for the sequences determined in this work is AJ238884.
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INTRODUCTION |
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During recent years, the function of the alternative sigma factor RpoS (38) has been extensively studied in Escherichia coli and other Gram-negative bacteria. RpoS is a regulator of stationary-phase-induced genes and is required for survival during stress and starvation (for reviews, see Hengge-Aronis, 1996
; Loewen & Hengge-Aronis, 1994
; Loewen et al., 1998
). Recently, the rpoS gene from Er. carotovora subsp. carotovora strain 71 was cloned and an rpoS mutant strain was constructed and characterized (Calcutt et al., 1998
; Mukherjee et al., 1998
). It was shown that RpoS is needed to withstand various environmental stresses in vitro, and that the rpoS mutant had increased maceration capability and produced increased amounts of extracellular enzymes in vitro. This effect was found to be mediated by lower expression of the negative regulator rsmA in the rpoS mutant (Mukherjee et al., 1998
). We have previously found that a mutant that overproduces RpoS due to a mutation in a gene designated expM has reduced virulence and is affected in extracellular enzyme production and secretion (Andersson et al., 1999
). The overproduction of RpoS was found to partly cause the phenotype of the expM mutant. The ExpM protein is a response regulator homologous to RssB/SprE in Es. coli and MviA in Salmonella typhimurium. These proteins belong to a new group of response regulators that are involved in the control of the stability of RpoS (Andersson et al., 1999
; Bearson et al. , 1996
; Muffler et al., 1996
; Pratt & Silhavy, 1996
). In this study we set out to characterize the phenotype of an rpoS mutant of Er. carotovora subsp. carotovora strain SCC3193, especially with regard to its virulence on tobacco plants. The mutant caused more severe symptoms than the wild-type strain on tobacco, presumably due to higher in planta production of extracellular enzymes, although it did not grow to higher cell densities on these plants. However, we show that the mutant was not able to infect tobacco with reduced catalase levels as efficiently as the wild-type strain, and that the growth of the mutant was impaired on these plants. Furthermore, we found that the rpoS mutant was outcompeted by the wild-type strain in planta and in vitro. As expected, we found that the rpoS mutant was more sensitive than the wild-type strain to osmotic and oxidative stress in vitro.
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METHODS |
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Nucleotide sequencing.
Nucleotide sequence of both strands of the Er. carotovora subsp. carotovora rpoS gene was determined by the dideoxy chain-termination method (Sanger et al., 1977 ) using customized synthetic primers (15- to 22-mers; GENSET OLIGOS) and Sequenase system (version II) from US Biochemicals with [
35 -S]dATP (Amersham Life Sciences). A BLAST search was used to compare the deduced amino acid sequence from the Er. carotovora subsp. carotovora rpoS gene and RpoS proteins from other bacteria.
Glycogen production, catalase assays and Erwinia autoinducer (EAI) measurement.
Accumulation of glycogen was tested by growing cells overnight on agar plates containing 1% glucose and then staining with iodine vapour (Latil-Damotte & Lares, 1977 ). Catalase activity was assayed by measuring hydrogen peroxide decomposition in a spectrophotometer at 240 nm (Beers & Sizer, 1952
). The amount of EAI was determined by a bioassay; Er. carotovora subsp. carotovora strain SCC3065 (Pirhonen et al., 1991
, 1993
), unable to produce EAI and harbouring the pHV200I- plasmid, was mixed with various dilutions of supernatant from SCC3193 and SCC8002. Light production, and hence the concentration of EAI, was then measured in a luminometer.
Enzyme assays and virulence tests.
The activities of Peh, Pel, Prt and Cel in vitro were assayed as described previously (Pirhonen et al., 1991 ). The levels of Peh, Pel and Prt in planta were measured as follows. Twenty-four plants (Nicotiana tabacum cv. Xanthi) were infected for each strain (about 2x106 c.f.u. per plant). After 48 h incubation plants were pooled in groups of six and the fresh weight was determined. Plants were then homogenized in 1 ml water. Plant material and bacteria were removed by centrifugation and the extracellular enzyme activities were determined. The results are expressed as activity per gram fresh weight (units for Peh, 0·1 µmol glucose equivalent min -1; for Prt, increase in A436 h -1 (ml sample)-1; and for Pel, A 548). As a control, the enzyme activities in uninfected plants were determined. Symptom development and bacterial growth in planta were determined in wild-type plants (N. tabacum cv. Xanthi) and antisense catalase (Ascat1 no. 17) plants (Takahashi et al., 1997
). The plants were grown for 4 weeks at 22 °C in a growth chamber [16 h light regime, 150 µE s-1 m-2 (16·7 mlx)]. Plants were then infected and bacteria extracted essentially as described by Vidal et al. (1998)
with the exception that 23x106 c.f.u. per plant (equal numbers for SCC3193 and SCC8002) were used to infect plants. After infection, plants were incubated for 48 h under the same conditions as above, but at 100% humidity to allow efficient infection. The results were analysed statistically by means of the Students t-test for which P-values are reported. Symptom levels were determined according to the following scale: 0=no symptoms, 1=clear infection of one leaf, 2=one leaf macerated, 3=infection spread to more than one leaf, 4=several leaves macerated, 5=only a few leaves uninfected. The mean symptom level was then calculated by adding the symptom levels of the infected plants and dividing by the number of plants used in each experiment. Salicylic acid (SA) treatment was done by adding SA to a final concentration of 1 mM to the plant growth medium 24 h before infection. When competition between SCC3193 and SCC8002 was studied in planta, 15 plants (N. tabacum cv. Xanthi) were infected with a mixture of equal numbers of both strains (a total of 2·1x106 c.f.u. per plant). After incubation for 48 h, bacteria were extracted and serial dilutions were plated on L medium with and without Km. The total c.f.u. and the c.f.u. on Km plates (SCC8002) were calculated as c.f.u. per infected plant. As a control, 30 plants were infected by SCC3193 and SCC8002 alone (2·8x106 and 2·0x106 c.f.u. per plant, respectively). Virulence tests on potato stems were performed on greenhouse-grown potato (Solanum tuberosum cv. Bintje), using a toothpick to infect the plants directly into the stem as described by McMillan et al. (1993)
.
RNA isolation, Northern and Western blot analysis.
RNA for Northern blot analysis was isolated by the Qiagen RNA isolation kit. Samples were collected during 24 h of growth and Northern blots were performed as described previously (Andersson et al., 1999 ). Western blots with Es. coli RpoS antisera (Jishage & Ishihama, 1995
) were performed according to Andersson et al. (1999
).
Stress experiments and competition in vitro.
Stress experiments during stationary phase were performed as follows (repeated at least twice). The cells were inoculated into 5 ml L medium and grown for 16 h to stationary phase. To test osmotic stress resistance, the cells were pelleted and resuspended in fresh L medium (5 ml) with or without the addition of 1 M NaCl. The cells were then grown for 24 h at 28 °C. To test oxidative stress resistance, 10 mM H2O2 was added directly to an overnight culture grown for 16 h and the cells were incubated for 4 h at 28 °C. The cells were plated and counted before and after the stress treatments. Complementation of the salt stress sensitivity of Es. coli strain RH90 was done essentially as above. The Es. coli strain MC4100 and the plasmid pMMKatF2 were used as controls in these experiments. Competition experiments between SCC3193 and SCC8002 in vitro were done as follows. The strains were grown separately for 16 h. They were then mixed in equal numbers and plated on L medium with and without Km to determine the c.f.u. in the inoculum. The mixture was diluted ten times in 10 mM MgSO4 and 10 µl of this dilution (around 3x106 total c.f.u.) was used to inoculate 5 ml L medium. The culture tubes were then grown for 48 h and again plated on L medium with and without Km. As controls, the strains were inoculated separately using the same procedure. This experiment was done four times with similar results. For stress tests during exponential phase, an overnight culture was subcultured into 5 ml fresh L medium and grown to an OD600 of 1·0. At this point, the cells were pelleted and resuspended in 10 mM MgSO4 with or without the addition of 2·5 mM H2O2. The cells were then incubated for 10 min at room temperature and serial dilutions were plated. This experiment was done twice with essentially the same results.
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RESULTS AND DISCUSSION |
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Characterization of the stress tolerance of the Er. carotovora subsp. carotovora rpoS mutant in vitro
The sigma factor RpoS has been shown to be involved in the production of secondary metabolites, virulence and/or stress survival in many bacterial species, including plant and animal pathogens (Anderson et al., 1998 ; Fang et al., 1992
; Flavier et al., 1998
; Loewen et al., 1998
; Sarniguet et al., 1995
; Swords et al., 1997
; Wilmes-Riesenberg et al., 1997
). We therefore decided to investigate whether RpoS has similar functions in the Er. carotovora subsp. carotovora strain SCC3193. To determine whether RpoS is needed for stress tolerance in SCC3193 during stationary phase, we analysed the phenotype of SCC8002 under osmotic and oxidative stress in L medium (Table 2
). We found that SCC8002 was sensitive to these kinds of stresses as the survival of the rpoS mutant was significantly lower than for the wild-type strain. Es. coli rpoS mutants are deficient in the production of glycogen (Lange & Hengge-Aronis, 1991
). We therefore performed a glycogen plate assay and found that SCC8002 was affected in the production of glycogen (data not shown). We also performed a complementation test with pRA910 and found that the plasmid complemented the stress sensitivity of the mutant as well as the deficiency in glycogen accumulation (data not shown). The complementation of these phenotypes was not complete, but nevertheless evident, perhaps due to the low expression from the minor rpoS promoter. We also tested stress sensitivity during exponential growth (OD600 1·0; 3·34·5x108 c.f.u. ml-1 ). No significant difference in tolerance towards 2·5 mM H2O2 between SCC3193 and SCC8002 was shown as 14% of the cells survived for both strains. However, we found that the expM mutant strain SCC3032, which overproduces RpoS during exponential growth (Andersson et al., 1999
), had enhanced tolerance as 29% of the cells survived. The fact that the rpoS mutant did not show higher sensitivity than the wild-type might be explained by the low levels of RpoS found in the wild-type during exponential growth (Andersson et al., 1999
).
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Virulence of the rpoS mutant
To characterize the virulence of SCC8002 on plants, we performed an assay on in-vitro-grown tobacco. The rpoS mutant was found to cause more severe symptoms than the wild-type strain (see Fig. 2). This is in agreement with Mukherjee et al. (1998)
who showed that an rpoS mutant strain caused increased maceration of celery petioles. We also performed a virulence test on potato stems as described by McMillan et al. (1993 )
. The virulence of SCC8002 was found to be similar to SCC3193 (data not shown). The reason why the rpoS mutant appears to be more virulent on tobacco but not on potato is not known, but it may be due to the infection starting in different parts of the host plants (stem inoculation in potato, leaf inoculation in tobacco). However, our general experience is that the tobacco assay is more sensitive and recognizes smaller differences in virulence (R. A. Andersson & M. Pirhonen, unpublished).
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It has previously been shown that Ascat plants, as used in this study, are more resistant to tobacco mosaic virus (TMV) infection and that this effect is mediated through SA (Du & Klessig, 1997 ; Takahashi et al., 1997
). In tobacco, SA has been shown to induce resistance towards Er. carotovora subsp. carotovora (Palva et al., 1994
; Vidal et al., 1998
). To find out if SCC8002 was more sensitive to plant defence responses induced by SA and to rule out the possibility that the results obtained with the transgenic Ascat plants were due to higher levels of SA, we infected SA-treated tobacco plants with SCC3193 and SCC8002. On the control plants we found similar results as shown in Fig. 2
, whilst the SA-treated plants showed increased resistance at the level of both symptom development and bacterial growth in planta. We were not able to detect any significant difference in the growth of SCC3193 and SCC8002 on the SA- treated plants (data not shown). Taken together, our results suggest that SCC8002 is more sensitive to reactive oxygen species not only in vitro but also in planta. The results also suggest that the rpoS mutant is not affected in its ability to infect wild-type tobacco. On the contrary, it causes more severe symptoms than SCC3193 on wild-type tobacco plants. This suggests that the rpoS mutant produces more extracellular enzymes in planta. To investigate this, we performed enzyme assays by using macerated plant tissue to determine the in planta levels of Peh, Pel and Prt (Table 3
). We found that the level of Peh and Prt was significantly higher (twofold for Peh and sevenfold for Prt) in tobacco plants infected with the rpoS mutant as compared to plants infected with the wild-type strain, whilst the production of Pel did not differ between the two strains. It is therefore likely that the increased symptoms caused by the rpoS mutant strain are due to increased extracellular enzyme production in planta and not to higher cell density. This is in contrast with the in vitro results, as we could not find any difference in the levels of extracellular enzymes in vitro when the cells were grown in L medium with or without PGA. Whilst our work indicates that RpoS is a positive regulator of rsmA in SCC3193, it appears that the level of rsmA is more important in planta than during growth in L medium. This is supported by our previous results showing that the expM mutant strain SCC3032, which overproduces RpoS and thereby shows enhanced rsmA expression, has significantly lower virulence on tobacco than the wild-type strain (Andersson et al., 1999
). Introduction of an rpoS mutation in the expM mutant background caused an rsmA expression similar to that seen in an rpoS mutant and the expM rpoS double mutant was found to have almost fully restored virulence (Andersson et al., 1999
). In addition, the expM mutant is also affected in production and secretion of the extracellular enzymes in vitro (L medium plus PGA), but this phenotype seems not to be rsmA-dependent as the expM rpoS double mutant is similar to the expM mutant in this respect (Andersson et al., 1999
).
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ACKNOWLEDGEMENTS |
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Received 7 June 1999;
revised 3 September 1999;
accepted 10 September 1999.
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