Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Inactivation of the lpcC gene alters surface-related properties and symbiotic capability of Bradyrhizobium japonicum H.-I. Lee1,†, Y.-H. In1,2,†, S.-Y. Jeong1,2, J.-M. Jeon1, J.G. Noh2, J.-S. So2 and W.-S. Chang1 1 Department of Biology, University of Texas, Arlington, TX, USA 2 Department of Biological Engineering, Inha University, Incheon, Korea

Significance and Impact of the Study: This study demonstrates the role of the B. japonicum lpcC in nodulation with soybean and importance of cell surface hydrophobicity. The results also highlight that intact LPS is required for successful symbiosis between B. japonicum and soybeans. Our findings not only support previous studies emphasizing the necessity of LPS on the interaction between the two symbiotic partners, but also contribute to a better understanding of the symbiotic mechanisms.

Keywords biofilm, Bradyrhizobium japonicum, lipopolysaccharide, lpcC, motility, surface hydrophobicity, symbiotic nitrogen fixation. Correspondence Woo-Suk Chang, Department of Biology, University of Texas, Arlington, Texas 76019, USA. E-mail: [email protected] Jae-Seong So, Department of Biological Engineering, Inha University, Incheon 402-751, Korea. E-mail: [email protected] † These authors equally contributed to this work.

2013/2324: received 19 November 2013, revised 31 January 2014 and accepted 4 February 2014

Abstract We investigated the role of the Bradyrhizobium japonicum lpcC gene, encoding a mannosyl transferase, involved in the lipopolysaccharide (LPS) biosynthesis. The inactivation of the lpcC gene considerably altered the LPS structure and the cell surface properties. LPS analysis showed that the lpcC mutant JS715 had an abnormal LPS structure deficient in O-antigen. The cell surface hydrophobicity increased approximately threefold in JS715 compared to the wild type. The increased cell surface hydrophobicity is likely to be related with cell aggregation in the mutant culture. For the growth comparison, JS715 showed slower growth rate than the wild type. The motility of JS715 decreased in soft agar plates, but it showed enhanced biofilm-forming ability. Interestingly, JS715 was not able to nodulate the host legume soybean (Glycine max). This study shows not only that lpcC is involved in the biosynthesis of O-antigen in the B. japonicum LPS, but also that inactivation of the lpcC gene affects symbiotic capability of B. japonicum and surface-related properties such as cell hydrophobicity, biofilm formation and motility.

doi:10.1111/lam.12232

Introduction Bradyrhizobium japonicum fixes atmospheric nitrogen into ammonia in a symbiotic relationship with the host legume soybean (Glycine max). The symbiotic process between B. japonicum and the soybean is initiated by attachment of the bacterium to root hairs of the host plant, followed by root hair curling, infection thread formation and eventually forming nitrogen-fixing nodules (Stacey et al. 1991; Brechenmacher et al. 2010). The initiation of the symbiotic Letters in Applied Microbiology 59, 9--16 © 2014 The Society for Applied Microbiology

process is also regulated by nodulation (Nod)-factor signalling molecules produced by B. japonicum. As an intimate cell-to-cell interaction is necessary for this nodulation process, cell surface properties of the bacterium are of considerable importance (Park and So 2000). Rhizobial lipopolysaccharides (LPSs) are composed of a lipid A, a core oligosaccharide(s), and a polysaccharide chain known as O-antigen, but the structure varies depending on species, even among strains in a single species (Fraysse et al. 2003; Castro et al. 2008). There has 9

Bradyrhizobium japonicum lpcC

been evidence that intact LPSs are essential for the successful symbiosis between rhizobia and their legume hosts. Alteration in the O-antigen could result in failure of development of the infection threads or formation of pseudonodules in which infected bacteroids do not exist (Carlson et al. 1987; Cava et al. 1989). Some LPS mutants could nodulate their host plants eventually, although the nodulation was delayed; however, their nitrogenase activities in the nodules were significantly reduced, indicating that LPS can play an important role in the postinfection steps as well (Campbell et al. 2002; Vedam et al. 2004). The roles of rhizobial LPSs in symbioses between rhizobia and their host plants have been well reviewed by Becker et al. (2005) and Carlson et al. (2010). Furthermore, impaired LPS structure may alter rhizobial biofilm formation and/or motility (Vanderlinde et al. 2009; Lee et al. 2010), which is likely to influence interactions between the two partners (Fujishige et al. 2006) . In a previous Tn5 mutagenesis study of B. japonicum, a nodulation-defective mutant was shown to be deficient in O-antigen (Stacey et al. 1991). The Tn5-inserted 5
5-kb nucleotide fragment involved in LPS biosynthesis was subsequently cloned (So 1991), and within the cloned region, three open reading frames (ORFs) including rfaF (So et al. 2000), rfaD (Noh et al. 2002) and galE (Chang et al. 2008) encoding heptosyltransferase, heptose-6-epimerase and UDP-galactose-4-epimerase were characterized, respectively. Here, we extend our understanding of the 5
5-kb fragment involved in B. japonicum LPS biosynthesis by characterizing another ORF, lpcC, encoding a mannosyl transferase. lpcC is located between rfaF and galE, and its transcriptional direction is opposite to those of the two genes (Chang et al. 2008). In Rhizobium leguminosarum, mannosyl transferase is required for building the LPS core structure by providing a mannosyl linkage to the LPS precursor Kdo2-lipid IVA (Kanipes et al. 2003). In this study, we constructed an lpcC mutant of B. japonicum and examined its cell surface properties and symbiotic capability. We conclude that lpcC is another key gene to initiate the successful symbiotic relationship between B. japonicum and soybean plants.

H.-I. Lee et al.

band (LPS I) and a low molecular weight band (LPS II), while JS715 LPS displayed only LPS II (Fig. 1), which is an indicative of an incomplete LPS lacking O-antigen (Carlson et al. 1987; Priefer 1989; Chang et al. 2008). Disruption of lpcC causes inhomogeneous growth and slower growth rate In addition to LPS profiles, the disruption of lpcC might change several phenotypes of B. japonicum. One of the most interesting changes observed was the inhomogeneous growth. When cultivated in the liquid AG medium aerobically, the JS715 culture was inhomogeneous as the cells were clumpish and sank to the bottom of the culture tube (Fig. 2a). However, 61A101C and CS715 cells grew homogeneously in the same growth condition. The agglutinating behaviour of LPS mutant cells when grown in liquid media is common in rhizobia (Cava et al. 1989; Priefer 1989; Araujo et al. 1994). Additionally, JS715 showed longer lag phase to 36–48 h (Fig. 2b) and the lower final cell density compared to 61A101C and CS715 (Fig. 2b). Furthermore, generation time of JS715 (11
16  0
88 h) is longer than that of 61A101C and CS715 (8
37  0
12 and 8
12  0
18 h, respectively). O-antigen deficiency increases the cell surface hydrophobicity in JS715 In our previous study, the cell surface of an LPS-deficient mutant of B. japonicum, rfaF mutant, was more hydrophobic than that of the wild type (Park and So 2000). Likewise, JS715 showed approximately threefold increase in the cell surface hydrophobicity compared to 61A101C and CS715 (Fig. 3a). This seems to be related to the

1

2

3

LPS I

Results and discussion LPS II

Disruption of lpcC affects LPS profiles To reveal a role of lpcC in the LPS biosynthesis, we first constructed an lpcC mutant and its complementary strains. We anticipated that LPS of the lpcC mutant JS715 lacked the O-antigen because of incomplete mannosyl linkage in the core region. As expected, the LPSs extracted from the wild-type 61A101C and the complementary strain CS715 showed both a higher molecular weight 10

Figure 1 LPS profile of Bradyrhizobium japonicum 61A101C (wild type; Lane 1), the lpcC mutant JS715 (Lane 2) and the complementary strain CS715 (Lane 3). Samples were prepared by the standard extraction method (Carrion et al. 1990). Each lane contains 50 lg of LPS sample, which has been visualized on a Pro-Qâ Emerald 300-stained gel through SDS-PAGE (12% polyacrylamide). LPS I and LPS II represent bands with lower mobility and higher mobility, respectively.

Letters in Applied Microbiology 59, 9--16 © 2014 The Society for Applied Microbiology

H.-I. Lee et al.

Bradyrhizobium japonicum lpcC

(a)

(b) 1e+10

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*

*

*

*

1e+7

* * *

1e+6 1e+5 1

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40 60 Time (h)

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Figure 2 (a) Growth characteristics of the liquid cultures for the wild-type 61A101C (1), the lpcC mutant JS715 (2) and its complementary strain CS715 (3). Cultures were grown at 30°C for 4 days by shaking at 200 rpm. The yellowish colour of the complementary strain culture is due to tetracycline added in the culture. (b) Growth comparison among 61A101C, JS715 and CS715. Closed circles, open circles and closed triangles indicate 61A101C (●), JS715 (○) and CS715 (▲), respectively. Values are means  standard errors of the means for three experiments. Asterisks indicate that the CFU values for JS715 are significantly different from those for 61A101C or CS715 at each time point by Student’s t-test (P < 0
05).

inhomogeneous growth phenotype of JS715. The cell surface hydrophobicity increased by the absence of O-antigen might promote the autoaggregation of JS715 cells during the culture period. Lack of lpcC inhibits the motility of B. japonicum, but enhances biofilm formation As incomplete LPS can affect attachment (e.g. biofilm formation) and motility (Lee et al. 2010), we tested whether JS715 changed these characteristics. First, the soft agar (0
3%) assay showed that JS715 was significantly less motile than 61A101C (Fig. 3b). CS715 showed a similar wild-type phenotype, although it did not fully complement the motility phenotype (Fig. 3b); the diameters of the colonies are 22
2  0
6, 6
6  0
2 and 19
4  0
6 mm for 61A101C, JS715 and CS715, respectively. It is possible that the less motility may be due to the slower growth rate in the mutant. To check this possibility, we performed the control experiment using 1% solid agar plates. As shown in Fig. 3b, there was no difference in diameter among three strains, which rules out the possibility of an effect of growth rate on the motility assay. This result is consistent with our previous motility study for the rfaF mutant strain (Lee et al. 2010). Secondly, in the comparison of biofilm formation among the three strains, JS715 showed significantly more biofilm formation than 61A101C and CS715 after 7 days of incubation (Fig. 3c). Like motility phenotype, biofilm phenotype of JS715 is also consistent with the result for the rfaF mutant (Lee et al. 2010), suggesting that the enhancement of biofilm formation by JS715 is likely due to the increased cell surface hydrophobicity. Letters in Applied Microbiology 59, 9--16 © 2014 The Society for Applied Microbiology

lpcC is required for successful soybean nodulation by B. japonicum As many rhizobial LPS mutants have shown to be incapable of normally nodulating their host plants (Carlson et al. 1987; Cava et al. 1989), we tested the nodulation ability of JS715. The mutant did not form mature nodules on the soybean roots, while 61A101C formed 7
89  0
11 nodules per plant. Complementation of lpcC restored the nodulation ability of the mutant up to almost similar levels of the wild type (6
11  0
29 nodules per plant by CS715), indicating that lpcC plays an important role in the early stage of the symbiotic process between B. japonicum and soybeans. Taken together with the biofilm and motility results, it is possible that JS715 fails to nodulate the soybean roots because the mutant cells are not able to move towards the host plant. However, the previous studies on the relationship between motility and nodulation showed that Sinorhizobium meliloti mutants defective in motility or chemotaxis still generated normal nodules, although delayed nodulation was observed (Ames and Bergman 1981; Caetano-Anolles et al. 1988). Thus, motility may not be an important determinant of the symbiotic performance. In addition, an effect of motility on nodulation could not be assessed in the nodulation experiment carried out using plastic growth pouches, because bacterial population (ca. 1 9 108 cells ml1) could be saturating in this system. Alternatively, the autoaggregation of the mutant cells, presumably resulting from more hydrophobic nature of the mutant cell surface, is likely to affect the proper surface-to-surface contact between bacterial and plant cells that are required during host cell wall invagination and 11

Bradyrhizobium japonicum lpcC

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H.-I. Lee et al.

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Figure 3 (a) Cell surface hydrophobicity of the wild type (61A101C), lpcC mutant (JS715) and its complementary strain (CS715). The cell surface hydrophobicity was measured by microbial adhesion to hydrocarbon (MATH) assay. Hexadecane was used as the source of hydrocarbon. The % MATH on the y-axis indicates the ratio of bacterial cells migrating from hydrophilic layer to hydrophobic layer of hexadecane, which was calculated by the formula described in the Materials and Methods. Values are means  standard errors for three independent experiments. (b) Motility of 61A101C, JS715 and CS715 strains. The motilities were tested by spotting 5 ll (ca. 2
5 9 106 cells) of mid-log grown bacterial culture on the solid (1% agar; left panel) and the semi-solid (0
3% agar; right panel) agar plate of AG medium. (c) Quantification of biofilms formed by 61A101C, JS715 and CS715 strains. The relative biofilm formation was calculated by dividing the amount of crystal violet-bound biofilms (A595) by the optical density (OD600) of the cell culture after 7 days of incubation. Values are means  standard errors of the means for three experiments, each comprising six technical replications. In panels a and c, different alphabet letters indicate statistically significant difference by Student’s t-test (P < 0
05).

12

infection thread progression. Therefore, the appropriate cell surface property specifically rendered by LPS structure and function might be a key factor for nodule development and symbiotic nitrogen fixation. To achieve nodule organogenesis, successful rhizobial attachment to root hairs of the host plant might be important (Rodriguez-Navarro et al. 2007). However, the enhanced biofilm formation by JS715 is not positively correlated with the nodulation on soybean roots. Similarly, the B. japonicum rfaF mutant showed nodulation-impaired, but biofilm-enhanced phenotype (Lee et al. 2010). It has been also reported that R. leguminosarum nodulates the host plants regardless of its biofilm-forming ability (Vanderlinde et al. 2009). In addition, there was an inverse relationship between nodulation and biofilm formation when B. japonicum was pretreated with coumestrol: the pretreatment induced more nodules than the untreated control, but its biofilm formation was inhibited by coumestrol (Lee et al. 2012). Therefore, the biofilm-forming ability is unlikely to be proportional to the nodulation ability. It is more likely that biofilms formed by rhizobia may function as a survival strategy in soils rather than a part of the nodulation process (Perez-Gimenez et al. 2009). To sum up, we showed altered characteristics of the lpcC mutant such as increased cell hydrophobicity, enhanced biofilm formation, but decreased motility and incomplete LPS, which ultimately led to defective nodulation on soybean roots. A mannosyl transferase encoded by lpcC is a key enzyme for intact LPS structure, presumably being involved in a mannosyl linkage of core region in B. japonicum. Our findings also support the importance of intact LPS in the rhizobium–legume symbiosis. However, we are still unaware of a molecular mechanism by which LPS is modulated to establish the successful symbiotic association between legumes and their symbionts. Rhizobia may produce LPS to protect themselves against host cell defence reactions such as secondary cell wall modification and sporadic cell death during the infection process (Perotto et al. 1994). On the other hand, the role of rhizobial LPSs could be as a nodulation signal molecule, although it has been almost canonical that Nod factors modulate the nodulation. LPS molecules from Rhizobium species are known to be able to promote the formation of infection threads on the white clover roots with a host-specific feature. It was reported that LPSs released from R. leguminosarum bv. Trifoli increased the infection, while those from nonspecific strains, R. leguminosarum bv. Viciae and Rhizobium meliloti, did not (Dazzo et al. 1991). In addition to the role of LPSs, it will be interesting to see in future studies whether the lpcC mutation affects production of Nod factors, exopolysaccharide (EPS) and capsular polysaccharide (CPS), all of which are likely to influence the infection process.

Letters in Applied Microbiology 59, 9--16 © 2014 The Society for Applied Microbiology

H.-I. Lee et al.

Bradyrhizobium japonicum lpcC

Materials and methods Bacterial strains and growth conditions The bacterial strains and the plasmids used are listed in Table 1. Escherichia coli strains were grown aerobically at 37°C in Luria-Bertani (LB) medium. B. japonicum strains were grown aerobically at 30°C in AG medium (Sadowsky et al. 1987). In the biofilm assay, B. japonicum strains were grown in Bergersen’s minimal medium supplemented with 0
4% (v/v) glycerol (BMM) (Bergersen 1961). Antibiotics and concentrations (lg ml1) used were as follows: chloramphenicol, 15 or 30; kanamycin, 50 or 150; gentamycin, 15 or 30; and tetracycline, 10 or 30 for E. coli or B. japonicum, respectively. Construction of an lpcC mutant and its complementary strain A 2
1-kb gene fragment containing lpcC was amplified from B. japonicum 61A101C genomic DNA by PCR using Table 1 Bacterial strains and plasmids

Strain/Plasmid

Relevant characteristics

Brdyrhizobium japonicum strains 61A101C Wild type; CmR JS715 61A101C with DlpcC::Km CS715 JS715 with pCS7 Escherichia coli strains DH5a supE44, DlacU169 (Φ80 lacZDM15) hsaR17 recA1 endA17 gyrA96 thi-1 relA1 S17-1 recA pro (RP4-2Tet::Mu Kan::Tn7) Plasmids pKD4 Template for amplifying kanamycin resistance gene; KmR pJQ200SK GmR; sacB pJ07 pJ2K7 pLAFR3 pLps pCS7

pJQ200SK carrying the 2
1 kb fragment of lpcC pJ07 containing DlpcC::Km pLAFR1 containing HaeII fragment of pUC8 pRK415 clone carrying the 5
5-kb EcoRI LPS fragment, TcR pLAFR3 carrying the 2
2 kb lpcC region derived from PstI/BamHI fragment of pLps

Reference or source

Nitragin, Wiscosin This study This study

the primers, LPC1F (50 -TGTTAGGATCCAACGCTGGA CCTATT-30 ) and LPC1R (50 -ATATAGGGCCCAGCATGA TCTCGGTC-30 ), which have restriction enzyme recognition linkers for BamHI (LPC1F) and ApaI (LPC1R), respectively, at the 50 ends. The BamHI/ApaI-restricted PCR product was inserted into BamHI/ApaI-restricted pJQ200SK (Quandt and Hynes 1993), resulting in pJ07. The lpcC gene in pJ07 was disrupted by inserting kanamycin resistance gene from pKD4, resulting in pJ2K7. pJ2K7 was introduced to E. coli S17-1, in which the conjugal transfer function is integrated (Simon et al. 1983). As pJQ200SK is not replicable in B. japonicum and is sensitive to sucrose due to sacB gene, we employed the positive selection in which single-homologous recombination events were eliminated. The lpcC mutant created by double-homologous recombination was selected on AG agar plates containing chloroamphenicol (30 lg ml1), kanamycin (150 lg ml1) and 8% (w/v) sucrose. The mutant strain was named JS715. To restore the function of lpcC in the mutant, a complementary strain was constructed by inserting a 2
2-kb BamHI/PstI-restricted fragment containing lpcC, derived from pLps (So et al. 2000), into pLAFR3 (Staskawicz et al. 1987), resulting in pCS7. Then, pCS7 was introduced into JS715 by triparental mating. The complementary strain was named CS715. All restriction enzymes used were purchased from Fermentas (Glen Burnie, MD). The construction of the mutant and its complementary strain was confirmed by PCR (data not shown). LPS profile analysis

Invitrogen

Simon et al. (1983) Datsenko and Wanner (2000) Quandt and Hynes (1993) This study This study Staskawicz et al. (1987) So et al. (2000) This study

CmR, chloramphenicol resistant; KmR, kanamycin resistant; GmR, gentamycin resistant; and TcR, tetracycline resistant.

Letters in Applied Microbiology 59, 9--16 © 2014 The Society for Applied Microbiology

LPS samples were prepared from the cell pellets by the previously established method for the LPS extraction (Carrion et al. 1990). The LPSs were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The LPS samples (10 ll each) were loaded on a discontinuous acrylamide/bisacrylamide gel, which was run with the currents of 13 mA (stacking gel) and 25 mA (resolving gel) until the loading dye reached the bottom of the gel. Subsequently, the LPSs were visualized on a gel stained with Pro-Qâ Emerald 300 LPS gel stain kit (Molecular Probesâ, Eugene, OR). Cell surface hydrophobicity analysis To determine the cell surface hydrophobicity, we performed the microbial adhesion to hydrocarbons (MATH) assay (Rosenberg et al. 1980). Hexadecane (Fisher Scientific, Fair Lawn, NJ) was used as a hydrocarbon source. The bacterial cells were harvested at mid-log phase, washed with phosphate-buffered saline (PBS, pH 7
0) and resuspended in PBS to adjust OD600 = 1. The bacterial 13

Bradyrhizobium japonicum lpcC

suspension (2 ml) was mixed with 0
2 ml of hexadecane, incubated at 30°C for 10 min and vortexed for 5 s. After an additional incubation for 15 min at room temperature, the absorbance of the lower, aqueous layer was measured at 600 nm. The bacterial cell removal rate was determined by the following calculation: % MATH = {(A0–At)/A0} 9 100, where A0 is the initial absorbance of the bacterial culture and At is the absorbance of the aqueous layer removed after the reaction. Motility assay We employed the soft agar assay to determine the motility (Liu et al. 2005). Five microlitres (ca. 2
5 9 106 cells) of mid-log-phase bacterial suspension was spotted on both solid (1% agar) and semi-solid (0
3% agar) AG agar plates. Because of different CFU between the wild-type and mutant cells at mid-log-phase, appropriate dilutions or concentrations were performed to achieve the number of ca. 2
5 9 106 cells. The motility was quantified by measuring the diameter of the cell diffusion on the agar plates after incubation for 7 days at 30°C.

H.-I. Lee et al.

soybean nodulation. Soybean seeds were prepared and germinated as described in a previous study (Lee et al. 2012). Each seedling was aseptically transferred into autoclaved plastic growth pouches and inoculated with ca. 1 9 108 cells (1 ml of OD600 = 0
1). A total of 9 soybean plants were tested for each strain, and the experiment was repeated three times independently. The plants were grown at 26°C with light source operated on the cycle of 16-h-on and 8-h-off a day and were watered with halfstrength B&D medium which does not contain any combined nitrogen source (Broughton and Dilworth 1971). Nodule numbers were counted after 21 days postinoculation. Acknowledgements We thank Andrew J. Donati for his critical reading of this manuscript. This work was supported, in part, by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100016797) and an Inha University Research grant.

Biofilm assay

Conflict of Interest

The biofilm-forming ability was determined by the crystal violet staining method with a slight modification (Borucki et al. 2003). The bacterial cells were harvested at the midlog phase, washed and resuspended in BMM to adjust OD600 = 1. The bacterial suspension was diluted (0
1%, v/v) in BMM, transferred onto a 96-well microtitre plate (200 ll per well) with six technical replicates per each sample and incubated at 30°C for 7 days. Cell density was measured by reading absorbance at 600 nm (A600). After culture medium and planktonic cells were aseptically removed, each well was washed three times with sterile ddH2O. The biofilm cells attached on the inside wall of each well were stained with 200 ll of 0
1% (w/v) crystal violet by incubation at room temperature for 45 min in the dark. Unbound dye was removed by vacuum suction, and each well was gently washed three times with sterile ddH2O. To destain biofilm cells, 200 ll of 100% ethanol was added, and after resuspending, half (100 ll) was transferred to a new 96-well microtitre plate. Then, the absorbance was determined at 595 nm (A595). The relative biofilm formation was normalized by dividing the A595 by A600. Experiments were repeated three times.

No conflict of interest declared.

Nodulation assay A laboratory-scale nodulation assay using plastic growth pouches (Mega International, St. Paul, MN) was performed to investigate the effect of the lpcC gene on 14

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Caetano-Anolles, G., Wall, L.G., Demicheli, A.T., Macchi, E.M., Bauer, W.D. and Favelukes, G. (1988) Role of motility and chemotaxis in efficiency of nodulation by Rhizobium meliloti. Plant Physiol 86, 1228–1235. Campbell, G.R., Reuhs, B.L. and Walker, G.C. (2002) Chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti requires correct lipopolysaccharide core. Proc Natl Acad Sci USA 99, 3938–3943. Carlson, R.W., Kalembasa, S., Turowski, D., Pachori, P. and Noel, K.D. (1987) Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development. J Bacteriol 169, 4923–4928. Carlson, R.W., Forsberg, L.S. and Kannenberg, E.L. (2010) Lipopolysaccharides in Rhizobium-legume symbioses. In Endotoxins: Structure, Function and Recognition eds Wang, X. and Quinn, P.J. pp. 339–386. New York: Springer. Carrion, M., Bhat, U.R., Reuhs, B. and Carlson, R.W. (1990) Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum. J Bacteriol 172, 1725–1731. Castro, C.D., Molinaro, A., Lanzetta, R., Silipo, A. and Parrilli, M. (2008) Lipopolysaccharide structures from Agrobacterium and Rhizobiaceae species. Carbohydr Res 343, 1924–1933. Cava, J.R., Elias, P.M., Turowski, D.A. and Noel, K.D. (1989) Rhizobium leguminosarum CFN42 genetic regions encoding lipopolysaccharide structures essential for complete nodule development on bean plants. J Bacteriol 171, 8–15. Chang, W.S., Park, K.M., Koh, S.C. and So, J.S. (2008) Characterization of the Bradyrhizobium japonicum galE gene: its impact on lipopolysaccharide profile and nodulation of soybean. FEMS Microbiol Lett 280, 242–249. Datsenko, K.A. and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97, 6640–6645. Dazzo, F.B., Truchet, G.L., Hollingsworth, R.I., Hrabak, E.M., Pankratz, H.S., Philip-Hollingsworth, S., Salzwedel, J.L., Chapman, K. et al. (1991) Rhizobium lipopolysaccharide modulates infection thread development in white clover root hairs. J Bacteriol 173, 5371–5384. Fraysse, N., Couderc, F. and Poinsot, V. (2003) Surface polysaccharide involvement in establishing the rhizobium– legume symbiosis. Eur J Biochem 270, 1365–1380. Fujishige, N.A., Kapadia1, N.N., De Hoff, P.L. and Hirsch, A.M. (2006) Investigations of Rhizobium biofilm formation. FEMS Microbiol Ecol 56, 195–206. Kanipes, M.I., Ribeiro, A.A., Lin, S.H., Cotter, R.J. and Raetz, C.R.H. (2003) A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum – Purification, substrate specificity, and expression in Salmonella waaC mutants. J Biol Chem 278, 16356–16364.

Letters in Applied Microbiology 59, 9--16 © 2014 The Society for Applied Microbiology

Bradyrhizobium japonicum lpcC

Lee, Y.W., Jeong, S.Y., In, Y.H., Kim, K.Y., So, J.S. and Chang, W.S. (2010) Lack of O-polysaccharide enhances biofilm formation by Bradyrhizobium japonicum. Lett Appl Microbiol 50, 452–456. Lee, H.I., Lee, J.H., Park, K.H., Sangurdekar, D. and Chang, W.S. (2012) Effect of soybean coumestrol on nodulation ability, biofilm formation, and the transcriptional profile of Bradyrhizobium japonicum. Appl Environ Microbiol 78, 2896–2903. Liu, Y.Q., Gao, W.M., Wang, Y., Wu, L.Y., Liu, X.D., Yan, T.F., Alm, E., Arkin, A. et al. (2005) Transcriptome analysis of Shewanella oneidensis MR-1 in response to elevated salt conditions. J Bacteriol 187, 2501–2507. Noh, J., Kim, D.H., Oh, E.T. and So, J.S. (2002) Characterization of the rfaD gene region of Bradyrhizobium japonicum 61A101C. J Microbiol Biotechnol 12, 826–828. Park, K.M. and So, J.S. (2000) Altered cell surface hydrophobicity of lipopolysaccharide-deficient mutant of Bradyrhizobium japonicum. J Microbiol Methods 41, 219– 226. Perez-Gimenez, J., Mongiardini, E.J., Althabegoiti, M.J., Covelli, J., Quelas, J.I., Lopez-Garcia, S.L. and Lodeiro, A.R. (2009) Soybean lectin enhances biofilm formation by Bradyrhizobium japonicum in the absence of plants. Int J Microbiol 2009, 719367. Perotto, S., Brewin, N.J. and Kannenberg, E.L. (1994) Cytological evidence for a host-defense response that reduces cell and tissue invasion in pea nodules by lipopolysaccharide-defective mutants of Rhizobium leguminosarum strain 3841. Mol Plant Microbe Interact 7, 99–112. Priefer, U.B. (1989) Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae Vf39. J Bacteriol 171, 6161–6168. Quandt, J. and Hynes, M.F. (1993) Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127, 15–21. Rodriguez-Navarro, D.N., Dardanelli, M.S. and Ruiz-Sainz, J.E. (2007) Attachment of bacteria to the roots of higher plants. FEMS Microbiol Lett 272, 127–136. Rosenberg, M., Gutnick, D. and Rosenberg, E. (1980) Adherence of bacteria to hydrocarbons – a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9, 29–33. Sadowsky, M.J., Tully, R.E., Cregan, P.B. and Keyser, H.H. (1987) Genetic diversity in Bradyrhizobium japonicum serogroup-123 and its relation to genotype-specific nodulation of soybean. Appl Environ Microbiol 53, 2624–2630. Simon, R., Priefer, U. and Puhler, A. (1983) A broad host range mobilization system for in vivo genetic engineering

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– Transposon mutagenesis in Gram-negative bacteria. Nat Biotechnol 1, 784–791. So, J.S. (1991) Molecular cloning of a gene region from Bradyrhizobium japonicum essential for lipopolysaccharide synthesis. FEMS Microbiol Lett 83, 299–304. So, J.S., Kim, W.S. and Stacey, G. (2000) Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaF gene. FEMS Microbiol Lett 190, 109–114. Stacey, G., So, J.S., Roth, L.E., Bhagya, L.S.K. and Carlson, R.W. (1991) A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation. Mol Plant Microbe Interact 4, 332–340. Staskawicz, B., Dahlbeck, D., Keen, N. and Napoli, C. (1987) Molecular characterization of cloned avirulence genes from

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Race-0 and Race-1 of Pseudomonas syringae pv. Glycinea. J Bacteriol 169, 5789–5794. Vanderlinde, E.M., Muszynski, A., Harrison, J.J., Koval, S.F., Foreman, D.L., Ceri, H., Kannenberg, E.L., Carlson, R.W. et al. (2009) Rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility. Microbiology 155, 3055– 3069. Vedam, V., Haynes, J.G., Kannenberg, E.L., Carlson, R.W. and Sherrier, D.J. (2004) A Rhizobium leguminosarum lipopolysaccharide lipid-A mutant induces nitrogen-fixing nodules with delayed and defective bacteroid formation. Mol Plant Microbe Interact 17, 283–291.

Letters in Applied Microbiology 59, 9--16 © 2014 The Society for Applied Microbiology