Characterization of Esterase A, a Pseudomonas stutzeri A15 Autotransporter

Abstract

Autotransporters are a widespread family of proteins, generally known as virulence factors produced by Gram-negative bacteria. In this study, the esterase A (EstA) autotransporter of the rice root-colonizing beneficial bacterium Pseudomonas stutzeri A15 was characterized. A multiple sequence alignment identified EstA as belonging to clade II of the GDSL esterase family. Autologous overexpression allowed the investigation of several features of both autotransporter proteins and GDSL esterases. First, the correctly folded autotransporter was shown to be present in the membrane fraction. Unexpectedly, after separation of the membrane fraction, EstA was detected in the N-laurylsarcosine soluble fraction. However, evidence is presented for the surface exposure of EstA based on fluorescent labeling with EstA specific antibodies. Another remarkable feature is the occurrence of a C-terminal leucine residue instead of the canonical phenylalanine or tryptophan residue. Replacement of this residue with a phenylalanine residue reduced the stability of the β-barrel. Regarding the esterase passenger domain, we show the importance of the catalytic triad residues, with the serine and histidine residues being more critical than the aspartate residue. Furthermore, the growth of an estA-negative mutant was not impaired and cell mobility was not disabled compared to the wild type. No specific phenotype was detected for an estA-negative mutant. Overall, P. stutzeri A15 EstA is a new candidate for the surface display of proteins in environmentally relevant biotechnological applications.

Fig 1

(A) Schematic overview depicting the structural elements of EstA from P. stutzeri A15 and in mutated residues in the present study. (B) Multiple sequence alignment of characterized autotransporters belonging to clade II of the GDSL esterase family. The alignment was made using the CLUSTAL W algorithm in Geneious (11). Only the passenger domain and the α-helical linker domain are shown, as indicated in panel A. Conserved sequence blocks (I, II, III, IIIa, and V) for clade II of the GDSL family have been boxed and named (2). “△” and “○” symbols indicate the residues of the catalytic triad and oxyanion hole, respectively. The α-helical region is indicated by an “α” underneath the alignment. This region was determined based on the crystal structure of EstA from P. aeruginosa (46). Strains: PSEstA, Pseudomonas stutzeri A15 EstA (ABP80765); PAEstA, Pseudomonas aeruginosa PAO1 EstA (AAB61674); XVEstE, Xanthomonas vesicatoria EstE (AAP49217); PLLipI, Photorhabdus luminescens LipI (CAA47020); STApeE, Salmonella enterica serovar Typhimurium ApeE (AAC38796); SLEstA, Serratia liquefaciens EstA (AAO38760).

Fig 2

Amino acid sequence alignment of C-terminal residues of 41 OMPs of P. stutzeri A15. OMPs were detected with the BOMP server (4) and verified using HHOmp (30) with a cutoff value of 95%. Sequence patterns were constructed in Geneious (11). (A) Thirty-one OMPs containing the classical C-terminal signature sequence for OMPs with hydrophobic residues at positions 3, 5, 7, and 9 relative to the C-terminal residue. Apart from the classical phenylalanine or tryptophan residue (39), the C-terminal residue can be a leucine residue in P. stutzeri A15. (B) Ten OMPs of P. stutzeri A15 containing an alternative C-terminal signature sequence with hydrophobic residues at the positions 2, 4, 6, 8, 9, and 10 relative to the C-terminal residue. Again, the C-terminal amino acid can be a leucine residue.

Fig 3

Heat modifiability of EstA in the TM fraction of P. stutzeri A15(pHERD26T-estA) (A) and P. stutzeri A15(pHERD26T-estA L636F) (B). TM fractions were prepared for SDS-PAGE but incubated at different temperatures. After SDS-PAGE, Western blotting was performed with anti-EstA antibodies.

Fig 4

Sarcosyl solubility of EstA from P. stutzeri A15(pHERD26T-estA). Proteins of the total lysate (TL), the cytoplasmic and periplasmic (CPP) fraction, the total membrane (TM) fraction, the sarcosyl soluble fraction (SSF), and the outer membrane (OM) fraction were prepared by ultracentrifugation (see the text) and analyzed with SDS-PAGE. Subsequently, Western blotting was performed with anti-EstA (upper part) and anti-OmpA (lower part) antibodies. EstA and OprF are indicated with arrows.

Fig 5

Effect of sarcosyl on the TM fraction of P. stutzeri A15(pHERD26T-estA) (A) and P. stutzeri A15(pHERD26T-estA L636F) (B). The TM fraction was incubated with 0% (lane 1), 1.25% (lane 2), 2.5% (lane 3), 3.75% (lane 4), and 5% (lane 5) sarcosyl (final concentration) and analyzed by SDS-PAGE. Subsequently, Western blotting was performed with anti-EstA antibodies.

Fig 6

Effect of sarcosyl on untreated and denatured TM fractions of P. stutzeri A15(pHERD26T-estA). TM fractions were incubated 10 min at 70°C plus 2% SDS (final concentration) (Heat/SDS +) or at room temperature (Heat/SDS −). Hereafter, 5% sarcosyl (final concentration) was added (Sarcosyl +) or omitted (Sarcosyl −). Sample preparation for SDS-PAGE was done at 70°C (Inc. Temp. 70) or 50°C (Inc. Temp. 50). Subsequently, Western blotting was performed with anti-EstA antibodies.

Fig 7

Cell surface display of EstA, represented as frequency-based curves of FITC intensities of individual cells of P. stutzeri A15 (light gray diamonds), P. stutzeri A15(pHERD26T) (dark gray squares), and P. stutzeri A15(pHERD26T-estA) (black triangles). EstA was detected via primary polyclonal anti-EstA antibodies and secondary FITC-conjugated antibodies. PI was used to exclude cells with damaged membranes from the analysis.

Fig 8

Relative esterase activity of whole cells (open bars, lowercase) or total membrane fractions (gray bars, uppercase) of P. stutzeri A15(pHERD26T) (−), P. stutzeri A15(pHERD26T-estA) (EstA), P. stutzeri A15(pHERD26T-estA S37A) (S37A), P. stutzeri A15(pHERD26T-estA D304A) (D304A), and P. stutzeri A15(pHERD26T-estA H307L) (H307L), using p-nitrophenyl butyrate as a substrate. The data represent the means of three independent repeats ± 95% confidence intervals. The significance level (P < 0.05) as determined with a Student-Newman-Keuls test is indicated with a letter code.