Erwinia chrysanthemi O Antigen Is Required for Betaine Osmoprotection in High-Salt Media

Abstract

Cellular components necessary for osmoprotection are poorly known. In this study we show that O antigen is specifically required for the effectiveness of betaines as osmoprotectants for Erwinia chrysanthemi in saline media. The phenotype is correlated with the inability of rfb mutant strains to maintain a high accumulation level of betaines in hypersaline media.

All microorganisms have to adapt to fluctuations in the osmolarity of their environment. In response to elevated medium osmolarity, bacteria first accumulate potassium and glutamate (6), which are rapidly replaced by few organic solutes, called osmoprotectants (4). In the enterobacteria, this family of molecules includes betaines like glycine betaine (GB) and imino acids (proline, pipecolate, etc.) (4, 7, 13). Enterobacteria like Escherichia coli or Erwinia chrysanthemi adopt a similar response to hyperosmotic stress regardless of the nature of the osmoprotectant available in the medium, and these molecules are accumulated in the cell proportionally to the osmotic stress applied via two systems, ProP and ProU (2-4, 8, 28).

All osmoprotectants are considered to be functionally equivalent. Nevertheless, Gutierrez and Csonka (9) have shown that adk mutations affect GB but not proline osmoprotection in Salmonella enterica serovar Typhimurium, and a similar phenotype was also found in a gltBD fnr mutant of E. coli (21). Moreover, an E. chrysanthemi bspA mutant grew poorly in the presence of salt and betaines (but not proline) (24). These studies underline differences between the mechanisms of action of GB and proline during ionic stress and show that osmoprotection is a complex mechanism that could not be summarized by osmoprotectant accumulation. To gain insight into these mechanisms, we selected mutants whose growth was no longer restored by GB in high-salt medium.

E. chrysanthemi A1828 (11) was subjected to mutagenesis with transposon Tn5-B21 (23). Two mutants, named W91 and W96, formed colonies on M63 basal medium supplemented or not with 0.5 M NaCl but did not grow on plates containing 0.5 M NaCl and 1 mM GB.

The growth of A1828, W91, and W96 was analyzed in M63 medium containing increasing NaCl concentrations in the presence or absence of 1 mM GB. W91 and W96 do not exhibit increased osmosensitivity (Fig. 1A to C). The addition of GB improved the growth of all strains on 0.3 M NaCl medium (Fig. 1A and B). With more stringent constraints (Fig. 1C), GB restored the W91 and W96 growth rate in the first stages of exponential growth, but a premature cessation of growth was observed that was more drastic as the NaCl concentration increased.

FIG. 1.

Effect of GB, as a function of medium osmolarity, on the growth of A1828 and W91 cells cultivated in high-salt media. Wild-type strain A1828 (squares) and mutant strain W91 (circles) were grown on M63 medium (A) alone or containing 0.3 (B) or 0.5 (C, D) M NaCl in the absence (open symbols) or in the presence (filled symbols) of 1 mM GB. The influence of the carbon source concentration was analyzed (D) by growing the cells on M63-0.5 M NaCl-1 mM GB medium containing 10 (small symbols), 20 (medium symbols), or 30 (large symbols) mM glucose. Similar results were obtained with strain W96 in place of strain W91. OD, optical density.

Increasing the glucose concentration enhanced the growth yield of strain A1828 but not that of strain W91 (Fig. 1D). Therefore, the phenotype did not result from carbon source depletion.

When the medium osmolarity was increased with nonionic osmotic agents like 0.8 M sucrose (1,100 osmol/kg of H₂O), the growth of W91 and W96 was identical to that of parental strain A1828. Similar results were obtained when GB was added to the medium at 0.8 M, acting both as an osmotic agent (1,100 osmol/kg of H₂O) and as an osmoprotectant. Thus, GB is not toxic in media of high osmolarity. In contrast, in the presence of 0.5 M KCl, K₂SO₄, or sodium glutamate GB failed again to act as a potent osmoprotectant. These results clearly show that the GB⁻ phenotype is associated with a concomitant effect of GB and high salt concentrations.

Among the various osmoprotectants effective on E. chrysanthemi under hyperosmotic conditions (7), pipecolate, ectoine, and proline improved the growth of W91 and W96, whereas dimethylsulfonioacetate and dimethylsulfoniopropionate, like GB, were unable to alleviate the inhibitory effect of a high salt concentration on their growth yields.

The levels of intracellular GB and pipecolate contents of strains A1828 and W91 were analyzed (8) in media of increasing osmolarity (from 0.3 to 0.5 M NaCl) containing 1 mM [¹⁴C]GB or [¹⁴C]pipecolate. In the wild-type strain, the GB content increased proportionally with the medium osmolarity, yielding 1,000 ± 96, 1,300 ± 127, and 1,400 ± 41 nmol mg of dry weight (DW)⁻¹ at 0.3, 0.4, and 0.5 M NaCl, respectively. In contrast, the level of GB accumulated by the W91 strain remained constant at 900 ± 24 nmol mg of DW⁻¹ regardless of the medium osmolarity. This accumulation defect was not observed when 1 mM [¹⁴C]pipecolate was used as an osmoprotectant, since the levels reached at 0.5 M NaCl were 1,800 ± 200 and 1,900 ± 290 nmol mg of DW⁻¹ for the wild type and the mutant, respectively. On the other hand, the [¹⁴C]GB uptake of cells of the wild-type and mutant strains cultivated at 0.5 M NaCl did not exhibit any significant differences (25 ± 3 and 17 ± 4 nmol min⁻¹ mg of DW⁻¹, respectively). In conclusion, the mutants exhibit a defect in GB accumulation at high salt concentrations that cannot be assigned to an alteration of the uptake systems.

To identify the mutations, the genomic regions flanking the inserted transposon in both mutants were cloned and sequenced (see Fig. 2 for details) (accession number AF503594). These nucleotide sequences were aligned to the entire nucleotide sequence of the E. chrysanthemi 3937 genome (https://asap.ahabs.wisc.edu/annotation). This analysis revealed that the transposon in strains W91 and W96 is inserted into two different and contiguous genes (wzt and wzm, respectively) belonging to a locus of eight open reading frames with the same transcriptional orientation (Fig. 2A). Searches of the databases have shown considerable homologies with the O-antigen export and biosynthesis pathway genes (Fig. 2B). The gene nomenclature was chosen on the basis of those amino acid sequence homologies and in accordance with the nomenclature proposed by Reeves et al. (19) relative to bacterial polysaccharide synthesis.

FIG. 2.

Genetic organization of the E. chrysanthemi rfb locus. (A) Genetic organization of the E. chrysanthemi rfb locus, restriction map, and subcloning of the mutated region. The Tn5-B21 transposon is represented at the top, and the line below shows the mutated chromosomal region; the insertion points of the transposon in strains W91 and W96 are indicated by vertical arrows. The genes are represented by horizontal arrows, the direction of which indicates the transcriptional direction. Putative transcriptional terminators are indicated by Ω. Two additional mutations created by cassette insertion into the wbeA and gmd sequences, resulting in strains W54 and W141, respectively, are indicated by vertical arrows. The last three lines show the inserts of the plasmids carrying the cloned chromosomal junctions from mutants W91 and W96. (B) Putative pathway for the biosynthesis and assembly of E. chrysanthemi O antigen based on sequence homologies.

The lipopolysaccharides (LPSs) of both the wild-type and mutant strains were extracted and separated by sodium dodecyl sulfate-13.5% polyacrylamide gel electrophoresis and visualized by silver staining (10, 27). wzm and wzt mutants lacked the O-antigenic structure but were not altered in the electrophoretic mobility of the core component (data not shown). Moreover, these mutants are resistant to bacteriophage φEC2 (φEC2^r), which is known to adsorb to the O antigen of E. chrysanthemi (22).

Strains W54 (wbeA::Ω-Amp^r) and W141 (gmd::′uidA-Kan^r) were constructed, and they showed the same phenotype as W91 or W96 regarding the use of GB as an osmoprotectant and O-antigen production. These results suggest that the entire rfb locus is implicated in the GB⁻ phenotype.

EDTA is known to provoke a release of LPS by chelating divalent cations that are necessary to stabilize the LPS structure (18). The wild-type strain was grown in M63-0.5 M NaCl-1 mM GB medium supplemented or not with EDTA at a concentration of 50 or 100 μM, and proline in the place of GB served as a control. The results presented in Fig. 3 show that the addition of 50 μM EDTA had no effect on A1828 growth in hyperosmotic medium plus GB or proline. In contrast, GB, but not proline, osmoprotection was impaired in the presence of 100 μM EDTA, as observed for the rfb mutant strains grown in the same medium without EDTA. Thus, O antigen is directly implicated in the GB⁻ phenotype.

FIG. 3.

Effect of EDTA on the osmoprotective efficiency of GB and proline for E. chrysanthemi wild-type strain A1828 in high-salt medium. Strain A1828 was grown on M63-0.5 M NaCl medium containing 1 mM GB (open symbols) or 1 mM proline (filled symbols) in the absence (circles) or in the presence (triangles) of EDTA at 50 (A) or 100 (B) μM. OD, optical density.

Several studies have revealed a significant role for the O antigen in environmental stress adaptation (, 16, 17). LPS plays an essential role in outer membrane integrity, and its implication in the secretion, assembly, or folding of surface proteins was clearly demonstrated in vivo and in vitro (5, 20, 26). The E. chrysanthemi rfb mutants have a failure in the control of the internal GB pool under salt constraint, most likely because of a defect in the efflux systems, as the measured influx activities were correct. Osmotic adaptation involves numerous membrane proteins, whose function has not yet been elucidated (14, 15); therefore it is possible that, as for IcsA of Shigella flexneri (25) and Tcp of Vibrio cholerae O1 (12), O antigen influences the activity of proteins involved in the control of the internal GB pool.