Phylogenetic relationships within the genus Pseudomonas were examined by comparing partial (about 1000 nucleotides) rpoB gene sequences. A total of 186 strains belonging to 75 species of Pseudomonas sensu stricto and related species were studied. The phylogenetic resolution of the rpoB tree was approximately three times higher than that of the rrs tree. Ribogroups published earlier correlated well with rpoB sequence clusters. The rpoB sequence database generated by this study was used for identification. A total of 89 isolates (79.5%) were identified to a named species, while 16 isolates (14.3%) corresponded to unnamed species, and 7 isolates (6.2%) had uncertain affiliation. rpoB sequencing is now being used for routine identification of Pseudomonas isolates in our laboratory.

A study of representatives of the bacterial genus Pseudomonas, analysing a combined data set of four molecular sequences with completely different properties and evolutionary constraints, is reported. The best evolutionary model was obtained with a hierarchical hypothesis testing program to describe each data set and the combined data set is presented and analysed under the likelihood criterion. The resolution among Pseudomonas taxa based on the combined data set analysis of the different lineages increased due to a synergistic effect of the individual data sets. The unresolved fluorescens lineage, as well as other weakly supported lineages in the single data set trees, should be revised in detail at the biochemical and molecular level. The taxonomic status of biovars of P. putida is discussed.

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.

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.

A multiplex PCR based on oprI and oprL, coding for the outer membrane lipoprotein I and the peptidoglycan-associated lipoprotein OprL, respectively, was developed for the detection of Pseudomonas strains from a bacterial collection isolated from a small river. To study the diversity of these Pseudomonas isolates, an oprI-oprL gene sequence database of 94 Pseudomonas type strains was constructed. Phylogenetic analysis of the concatenated oprI and oprL gene sequences of the Pseudomonas type strains showed that they were largely congruent with the classification based on the MLSA approach based on 16S rRNA, gyrB, rpoB and rpoD gene sequences of Mulet et al. in 2010. Identification of the isolates demonstrated a high diversity of Pseudomonas isolates at the source of the river located in a forest of which most isolates belonged to the Pseudomonas fluorescens lineage. On the other hand, the Pseudomonas population isolated at an anthropized site at the mouth of the river, receiving waste water from both households and industry, was very different and contained many Pseudomonas aeruginosa isolates.