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. 1998 Dec;180(24):6551–6556. doi: 10.1128/jb.180.24.6551-6556.1998

Sequence Diversity of the oprI Gene, Coding for Major Outer Membrane Lipoprotein I, among rRNA Group I Pseudomonads

Daniel De Vos 1,†, Christiane Bouton 1, Alain Sarniguet 2, Paul De Vos 3, Marc Vauterin 4, Pierre Cornelis 1,*
PMCID: PMC107757  PMID: 9851998

Abstract

The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.

Pseudomonads are increasingly being recognized as important microorganisms in our biosphere, and Pseudomonas aeruginosa and Pseudomonas fluorescens are two important representatives of this genus. As a typical opportunist, P. aeruginosa is more and more involved in a variety of often fatal nosocomial infections, in which it accounts for more than 11% of all isolates recovered (29). In cystic fibrosis, one of the most common autosomal recessive genetic diseases, it is a characteristic pathogen responsible for most of the cases of morbidity and mortality (16, 38). In general, fluorescent pseudomonads, including P. aeruginosa, Pseudomonas putida, P. fluorescens, and other species, are frequently found as rhizosphere microorganisms, in some cases promoting plant growth (11, 19, 20).

P. fluorescens and P. aeruginosa are also found as inherent flora of mineral water (14, 39). Identification of fluorescent pseudomonads is often tedious and not reliable. Indeed, the present taxonomy of this group is far from clear at the finer taxonomic level, as polyphasic investigations have demonstrated (4, 13, 18, 26). Ribosomal RNAs have been applied as molecular markers with great success to unravel the rough phylogenetic structure which, at the finer level, is not always in complete agreement with the genotypic and phenotypic similarities deduced from other parts of the genome. Horizontal gene transfer, chromosomal mutation hot spots, and internal genomic rearrangements are probably the bases of these discrepancies at the species and subspecies levels. These arguments, together with the importance of discriminating phenotypic tests in routine identifications, support a polyphasic approach in bacterial taxonomy (2, 8–10, 13, 37, 40). Additional phylogenetic information requires the identification of molecules, like the recA or the gyrB genes, that are widely distributed, large enough to contain a substantial amount of information, and conserved to an appropriate degree (24, 46). In the phylogenetic tree published by Woese (43), species with the same generic name were allocated in phylogenetically distant groups. This was the case for the “genus” Pseudomonas, which is known to be a dump of assemblages of distantly related species (3, 8–10, 17). Taxonomic rearrangements of the genus Pseudomonas sensu stricto resulted in the splitting of the genus and as a logical consequence, the present genus Pseudomonas is restricted to the rRNA group I organisms, with P. aeruginosa as the type species in this group (27, 28, 37, 42, 44, 45).

The oprI gene, coding for the outer membrane lipoprotein I of P. aeruginosa (5), was found to be conserved among the fluorescent pseudomonads and was considered to be a possible phylogenetic marker (6, 31). In this study, we tested whether the oprI gene could be a useful detection and identification target molecule as well as a complementary phylogenetic marker for rRNA group I pseudomonads. Also, we examined to what extent the sequence variation of the oprI gene reflects the species diversity in P. aeruginosa and P. fluorescens.

MATERIALS AND METHODS

Strains and growth conditions.

The type and reference strains included in this study are listed in Fig. 1 and come from the collection of the Laboratorium voor Microbiologie (LMG; University of Ghent, Ghent, Belgium). In addition to these, 25 additional P. aeruginosa strains (22 clinical and 3 rhizosphere isolates) were also analyzed. All clinical isolates were provided by the University Hospital Erasme of Brussels (M. Struelens) or by the Burn Wound Center of Queen Astrid Military Hospital, Brussels, Belgium (A. Vanderkelen). Three pyoverdin-negative strains were received from Université Louis Pasteur, Strasbourg, France (J. M. Meyer). One rhizosphere isolate (7NSK2) was obtained from M. Höfte (University of Ghent), and the two others were isolates from chickpea (PNA1) or chili (SA44) roots. Twenty additional P. fluorescens strains were isolated from a wheat rhizosphere at INRA (Institut National de Recherche Agronomique) in Le Rheu, France. These isolates (designated Flur) were biochemically characterized by the following criteria: liquefaction of gelatin (production of gelatinase), utilization of trehalose, production of levan, denitrification, and utilization of l-arabinose and l-tartrate. On the basis of this characterization, the isolates were assigned to a specific biotype, as has been done by others (21, 22, 27).

FIG. 1.

FIG. 1

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Sequence comparison of the 249-bp open reading frame of oprI from P. aeruginosa PAO1 (ATCC 15692; accession number M25761), P. mendocina (LMG 1223T; GenBank accession no. AF065948), P. oleovorans (LMG 2229T; accession no. AF065949), P. pseudoalcaligenes (LMG 6036; accession no. AF065950), P. alcaligenes (LMG 1224; accession no. AF065951), P. pseudoalcaligenes (LMG 5516; accession no. AF065952), P. putida (LMG 2257T; accession no. AF065953), P. cichorii (LMG 2162T; accession no. AF065954), P. syringae (LMG 1247T; accession no. AF065955), P. caricapapayae (LMG 2152; accession no. AF065956), P. fluorescens (Flur 3; accession no. AF065957), P. fluorescens (Flur 7; accession no. AF065958), P. fluorescens (Flur 16; accession no. AF065959), P. fluorescens (Flur 5; accession no. AF065960), P. fluorescens (Flur 12; accession no. AF065961), P. fluorescens (Flur 15; accession no. AF065962), P. fluorescens (Flur 8; accession no. AF065963), P. fluorescens (Flur 2; accession no. AF065964), P. fluorescens (Flur 9; accession no. AF065965), P. fluorescens (Flur 10, Flur 14, Flur 19, Flur 20; accession no. AF065966), P. fluorescens (Flur 1, Flur 4; accession no. AF065967), P. fluorescens (Flur 6; accession no. AF065968), P. fluorescens (LMG 5168; accession no. AF065969), P. corrugata (LMG 2172T; accession no. AF065970), P. fluorescens (LMG 5939; accession no. AF065971), P. fluorescens (Flur 11, Flur 17; accession no. AF065972), P. fluorescens (Flur 13; accession no. AF065973), P. fluorescens (Flur 18; accession no. AF065974), P. fluorescens (ATCC 17400; accession no. AF065975), P. fluorescens (LMG 5938; accession no. AF065976), P. fluorescens (LMG 1244; accession no. AF05977), P. fluorescens (LMG 5833; accession no. AF065978), P. tolaasii (LMG 2342T; accession no. AF065979), P. chlororaphis (aureofaciens) (LMG 1245; accession no. AF065980), P. marginalis (LMG 2214; accession no. AF065981), P. agarici (LMG 2112T; accession no. AF065982), P. taetrolens (LMG 2336T; accession no. AF065983), and P. asplenii (LMG 2137T; accession no. AF065984). The nucleotides identical to those in the P. aeruginosa sequence are shown by a dot. The positions of the HaeIII, PvuII, and SphI sites are shaded. Sites shown in dark shading are actual restriction sites; others are lost, due to a nucleotide substitution.

Strains were grown in Luria-Bertani medium at 37°C (P. aeruginosa) or at 28°C (other Pseudomonas species).

Sequencing and data analysis.

Sequencing reactions were completed with the use of Autoload (Pharmacia-Biotech, Roosendaal, The Netherlands). This solid-phase sequencing kit is based on the classical dideoxy sequencing reaction using T7 DNA polymerase. Sequencing templates (PCR-amplified oprI genes) were obtained as described previously (6). One of the two primers was biotinylated at the 5′ end in order to immobilize the biotinylated PCR product onto a streptavidin-coated solid phase prior to the sequencing reaction with a fluorescein isothiocyanate-labelled primer. The oprI gene was always sequenced on both strands, once with the forward and once with the reverse primer. The sequencing gel was run on an automated sequencer (ALF DNA sequencer; Pharmacia-Biotech). The different oprI DNA sequences were analyzed for the presence of restriction sites and for their products and were aligned with the PCGENE (Intelligenetics) and GENECOMPARE (Applied Maths, Kortrijk, Belgium) software.

RESULTS

Restriction site analysis based on the oprI sequence of collection strains.

For the 25 strains of P. aeruginosa analyzed, single HaeIII, PvuII, and SphI sites were always located at positions 49, 112, and 228, respectively, along the 249-bp oprI coding sequence (Fig. 1). None of the strains belonging to the 16 other species of Pseudomonas rRNA group I included in this study showed a HaeIII site at position 49 with the exception of P. cichorii LMG 2162T, which had two HaeIII sites, at positions 37 and 49. Most oprI sequences showed a HaeIII site at position 37, either as a unique site or in combination with another site at position 31, 49, 127, or 169. One isolate had a single HaeIII site at position 31 (P. fluorescens Flur 2). In three sequences, no HaeIII site was found. When present, the unique PvuII site was always at position 112. The unique SphI site (at position 228) was found to be well conserved and was only absent in five sequences. None of the three restriction sites was detected in the P. putida LMG 2257T oprI sequence.

Sequence comparison.

The different oprI sequences were compared at the nucleotide level for the presence of substitutions, taking the P. aeruginosa PAO1 oprI sequence as the reference (Fig. 1). As already mentioned, the 25 sequences corresponding to P. aeruginosa isolates were extremely conserved: their sequence similarity after alignment was 99 to 100% (results not shown). Indeed, in the case of 11 P. aeruginosa isolates, only one silent substitution was observed, for a threonine codon (ACC→ACT) at position 96, as was previously observed during a comparison of three sequences of P. aeruginosa oprI (31). The other oprI sequences showed the presence of several mostly silent mutations and also some nonsilent substitutions, resulting in changes at the amino acid level as evidenced after alignment of the different OprI sequences (results not shown).

Only two nonsilent substitutions resulted in a change in the signal sequence of OprI. These were at residue 10, changing a leucine residue into an alanine (P. oleovorans LMG 2229T) or a valine (P. syringae LMG 1247T). The P. aeruginosa OprI sequence differed from all others at residue 23 since it was the only one to have a histidine at this position (a valine was found in 26 sequences, an alanine in 11, and a methionine in 5). Residue 41 (Ala in P. aeruginosa) was conserved only in seven sequences, while in all other sequences, it was replaced by a serine residue. Finally, the glycine residue at position 57 was found to be conserved only in the P. asplenii LMG 2137T sequence. Other substitutions were less frequently found, most of them clustered in the C-terminal part of the protein, between residues 68 and 76.

Gene diversity of oprI in fluorescent pseudomonads.

A classic UPGMA (unweighted pair group maximum average; CLUSTAL program, GENE COMPARE) dendrogram (15) (Fig. 2) shows that the P. aeruginosa sequences cluster, forming the most homogenous group, while P. fluorescens isolates are more scattered along the dendrogram. Some sequences were identical, e.g., those of P. alcaligenes LMG 1224 and P. pseudoalcaligenes LMG 6036 and those of P. chlororaphis T9 (a rhizosphere isolate [results not shown]) and the former type strain of P. aureofaciens LMG 1245. Other sequences clustered together, such as P. mendocina LMG 1223T and P. oleovorans LMG 2229T (98% similarity), P. cichorii LMG 2162T and P. syringae LMG 1247T (97% similarity), or P. fluorescens LMG 5833 and P. tolaasii LMG 2342T (99% similarity). Some wheat rhizosphere isolates showed identical oprI sequences. This was the case for Flur 3 (with a biotype phenotypically intermediary between P. fluorescens biovar I and P. putida) and 7 (regarded as P. fluorescens biovar III), Flur 5 and 16 (both P. fluorescens biovar V), Flur 12 and 15 (both P. fluorescens biovar III), Flur 10, 14, 19, and 20 (P. fluorescens biovar IV for Flur 10, P. fluorescens biovar II for the others), and Flur 11 and 17 (both P. fluorescens but without clear biovar assignment).

FIG. 2.

FIG. 2

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Sequence similarity tree (UPGMA) based on the comparison of the oprI nucleotide sequences shown in Fig. 1.

DISCUSSION

This study confirms that the oprI gene is a suitable additional marker for the molecular taxonomy of rRNA group I pseudomonads (6, 31). Indeed, the results presented here show that the lipoprotein oprI gene may show some degree of DNA sequence variation (frequent silent mutations) while its gene product, the OprI lipoprotein itself, is better conserved (few nonsilent mutations). As expected, the silent substitutions occurred often at the third position in the codon. All P. aeruginosa oprI sequences that were examined were highly conserved, with only one silent substitution at position 96. In general, based on restriction analysis, a rather unexpected grouping at the fine level was obtained. This is obviously best deduced from restriction site similarities found in the P. fluorescens collection strains. Indeed, P. fluorescens LMG 1244 and LMG 5168 show the same restriction pattern although they belong, respectively, to P. fluorescens biovars III and IV and are clearly differentiated in Biolog, Biotype 100, and ribotyping analyses. The same is true for P. fluorescens LMG 5833 and LMG 5938 (belonging to P. fluorescens biovars V and III, respectively), and also for P. fluorescens LMG 5939 (P. fluorescens biovar IV) and P. chlororaphis LMG 1245 (P. fluorescens biovar IV). Because of the very limited number of restriction sites, the discrepancies with other techniques is easily explained by the large impact of a difference in a single restriction site. On the contrary, the grouping obtained by sequence analysis is far more reliable. In the tree (Fig. 2), based on the sequence similarity of the lipoprotein I gene, P. aeruginosa forms a very sharply delineated and homogeneous species that is differentiated from the other fluorescent pseudomonads, a finding which is in agreement with previous work (1, 17, 25). In other species, the sequence variation is higher and, as in the case of P. fluorescens, it can be used to delineate subgroups. As a consequence of this observation, the sequence determined for P. aeruginosa can be considered a signature for this species. The high degree of conservation of the oprI sequence in P. aeruginosa is striking, given the fact that genomic rearrangements are known to occur frequently among representatives of this species, resulting in a mosaic genome structure (36). Our results also confirm that P. fluorescens is a heterogeneous species, compared to P. aeruginosa, since the oprI sequences corresponding to the different representatives of this species do not cluster together in the similarity tree (Fig. 2) but are rather spread out. Nonsilent substitutions, resulting in an amino acid change, were found to occur often at some preferred positions, but none of them affect the recognition of the lipoprotein by a monoclonal antibody against OprI (6). P. chlororaphis T9 (a sugar beet rhizosphere isolate from Belgium) and P. chlororaphis LMG 1245 (former type strain of P. aureofaciens) were found to cluster close together, a finding which correlates well with that of a previous study (17), while the clustering of the phytopathogens P. cichorii LMG 2162T and P. syringae LMG 1247T is also in agreement with the same study. P. pseudoalcaligenes LMG 5516 and LMG 6036, P. mendocina LMG 1223T, and P. oleovorans LMG 2229T are closer to P. aeruginosa according to our clustering, which is certainly in agreement with rRNA sequence data (28). Previous studies on the diversity of the rhizosphere pseudomonads from two plants, flax and tomato, have demonstrated that both the plant and the nature of the soil could influence the composition of the microflora (21, 22). In both studies, the most frequently identified biovars for P. fluorescens were biovars II and III, while it has been suggested that biovar V is the most prevalent in the rhizosphere of wheat in Australian soils (32). In conclusion, we suggest that oprI gene diversity fingerprinting (restriction analysis and SPS sequencing) can be useful, in addition to selective techniques to amplify and sequence 16S RNA (41), for the study of the diversity and dynamics of P. fluorescens rhizosphere populations over time and in relation to competitors such as phytopathogenic fungi (11, 33–35). Using a similar approach, it would also be interesting to compare the sequences of oprL, the gene for the peptidoglycan-associated lipoprotein, already described in P. putida (30) and P. aeruginosa (7, 23), in different rRNA group I pseudomonads.

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