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Departament de Biologia, Microbiologia, Universitat de les Illes Balears, Campus UIB, 07122 Palma de Mallorca, Spain,1 Institut Mediterrani d'Estudis Avançats (CSIC-UIB), Campus UIB, 07122 Palma de Mallorca, Spain,2 Department of Biochemistry and Microbiology, Rutgers University, Cook Campus, New Brunswick, New Jersey 08901-85203
SUMMARY INTRODUCTION DEFINITION OF THE SPECIES AND DIFFERENTIATION FROM OTHER PSEUDOMONAS SPECIES Definition Differentiation from Other Species DISCOVERY AND NOMENCLATURAL PROBLEMS OCCURRENCE AND ISOLATION PROCEDURES PHENOTYPIC PROPERTIES Colony Structures/Types Morphological Characterization (Cells, Reserve Materials, Flagella, and Pili) and Chemotaxis Chemical Characterization and Chemotaxonomy DNA base composition. Protein patterns. LPS and immunological characteristics. Fatty acid composition. Quinone and polyamine composition. PHA. GENOMIC CHARACTERIZATION AND PHYLOGENY DNA-DNA Hybridizations Genome Size and Organization Genotyping Genetic Diversity: MLEE Genetic Diversity: MLST Phylogeny Clonality TAXONOMIC RANKS: GENOMOVARS IDENTIFICATION Phenotypic Identification Molecular DNA-Based Identification Polyphasic Identification PHYSIOLOGICAL PROPERTIES Temperature, Pressure, pH, and O2 Relationships Denitrification Structural gene clusters and the nature of denitrification genes. (i) nar genes. (ii) nir genes. (iii) nor genes. (iv) nos genes. Metalloenzymes involved in the denitrification process. (i) Nitrate respiration and NaRs. (ii) Properties of NarL and NarX proteins. (iii) Nitrite respiration and NiRs. (iv) Nitric oxide respiration and NORs. (v) Nitrous oxide respiration and N2ORs. Chlorate and Perchlorate as Terminal Electron Acceptors Organic Compounds Used as the Sole Carbon and Energy Source Inorganic Energy Sources (Thiosulfate) Production of Siderophores Nitrogen Fixation Phosphite and Hypophosphite Oxidation Biodegradation and Useful Properties for Biotechnological Applications Metal cycling. Crude oil, oil derivatives, and aliphatic hydrocarbons. Aromatic hydrocarbons. Biocides. Proteolytic activity: applications for biorestoration. NATURAL TRANSFORMATION PATHOGENICITY AND ANTIBIOTIC RESISTANCE HABITATS AND ECOLOGICAL RELEVANCE Soil, Rhizosphere, and Groundwater Marine Water and Sediment and Salt Marshes Wastewater Treatment Plants CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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| DEFINITION OF THE SPECIES AND DIFFERENTIATION FROM OTHER PSEUDOMONAS SPECIES |
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The species most closely related to P. stutzeri is P. balearica (formerly genomovar 6 of the species). It shares many basic phenotypic traits with P. stutzeri strains and belongs to the same 16S rRNA phylogenetic branch. However, it can be differentiated chemotaxonomically from P. stutzeri by its ability to grow above 42°C and by a few other biochemical tests (23).
P. chloritidismutans is a member of genomovar 3. However, it has been proposed as the type strain of a new species (404) and is discussed below (see "Physiological properties"). There is always a danger of drawing taxonomic conclusions from the properties of metabolic systems that are involved in the metabolism of unusual substrates or molecules.
The phylogenies of genes of the rrn operon, considered individually or with other housekeeping genes, demonstrate that all P. stutzeri strains are monophyletic. Such phylogenetic studies are currently another good tool for discriminating P. stutzeri from the rest of the bacterial species. P. xanthomarina has recently been described as a new species (289) with only one representative strain. It is located in the same 16S rRNA phylogenetic branch as P. stutzeri and P. balearica, with sequence similarities above 98%. It can be differentiated phenotypically from both species.
| DISCOVERY AND NOMENCLATURAL PROBLEMS |
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The type strain is Lautrop strain AB 201 (equivalent to Stanier 221, ATCC 17588, CCUG11256, DSM 5190, ICMP 12591, LMG11199, NCIB 11358, and WCPPB 1973). In addition, a reference strain has been proposed for each genomovar (Table 1). Some relevant strains that were previously assigned to other species are Pseudomonas perfectomarina strain ZoBell (19), Alcaligenes faecalis A15 (380), and Flavobacterium lutescens strain ATCC 27951 (24). Many, but not all, strains have been deposited in publicly recognized culture collections, are available for scientific research, and should be used as reference strains.
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| OCCURRENCE AND ISOLATION PROCEDURES |
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An elective culture method for the specific enrichment of denitrifiers and the isolation of P. stutzeri was developed by Iterson in 1902 (described in 1952 by van Niel and Allen [371]). A mineral medium with 2% nitrate under anaerobic conditions and tartrate (or malate, succinate, malonate, citrate, ethanol, or acetate) leads to a predominant population of P. stutzeri, even when some isolates are not able to grow on tartrate in pure culture. Tartrate may be converted anaerobically to an assimilable substrate by other bacteria in the sample. A selection of cells producing colonies with the unusual morphology of P. stutzeri permits an efficient isolation procedure from environmental samples. Incubation temperatures of 37°C or above allow a more selective enrichment, which can be combined with denitrifying conditions.
DNA methods based on 16S rRNA sequences have been also designed to detect P. stutzeri in DNA extracted directly from environmental samples. Bennasar et al., in 1998, developed PCR primers that were specific to all known genomovars of P. stutzeri at that time (24). This served as a confirmation test, as did amplicon cleavage using the restriction enzyme HindIII or a specific DNA probe targeted at the amplified product (24). Amann et al. considered the difficulty of obtaining a DNA probe to cover all of the P. stutzeri strains (5). However, they designed a DNA probe for specific 23S rRNA sequences. This is useful in fluorescence in situ hybridization techniques to detect and quantify P. stutzeri in environmental samples. Nevertheless, not all strains can be detected, due to the high genetic diversity of the species, including the rrn operon.
Besides the rrn genes, other genes are now used for functional analysis of ecosystems. These genes also detect P. stutzeri. They include nirS or nosZ for detecting denitrification (46) and nifH for analyzing diazotrophic bacteria in the rhizosphere (93). The usefulness of a conserved nosZ probe for screening the distribution of denitrifying bacteria with similar N2O reductases in the environment has been described elsewhere (65, 386). In 2001, Grüntzig et al. developed a very sensitive method based on real-time PCR analysis of DNA isolated from soil and sediment samples (132). However, not all DNAs of the species' strains could be amplified. Specific primers for PCR and an internal probe of the denitrification gene nirS enabled less than 100 cells per g of sample to be quantified.
In their analysis of P. stutzeri populations in marine waters, Ward and Cockcroft used monoclonal antibodies raised against outer membrane proteins of the strain ZoBell (388). ZoBell originally named this strain "Pseudomonas perfectomarina."
Sikorski et al. were able to isolate members of P. stutzeri from aquatic habitats and terrestrial ecosystems in a two-step procedure. Firstly, the occurrence of P. stutzeri cells was assessed by a previously designed, slightly modified PCR procedure (24, 325). Secondly, the positive samples were screened for P. stutzeri by means of plating on an artificial seawater medium with ethylene glycol, starch, or maltose as the carbon source under aerobic conditions (325). The characteristic colony morphology of P. stutzeri led to a highly efficient isolation procedure: one P. stutzeri colony was detected among 9,100 colonies of other bacteria.
However, many strains of P. stutzeri that have been studied in detail were isolated by their metabolic peculiarities. They were not specifically isolated for denitrification ability or because P. stutzeri was the target of the study.
| PHENOTYPIC PROPERTIES |
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Strains of P. stutzeri, like most recognized Pseudomonas spp., can grow in minimal, chemically defined media, with ammonium ions or nitrate and a single organic molecule as the sole carbon and energy source. No additional growth factors are required. Some P. stutzeri strains can grow diazotrophically. This characteristic seems to be rare among the genus Pseudomonas. None of the strains tolerate acidic conditions: they do not grow at pH 4.5. P. stutzeri has a respiratory metabolism, and oxygen is the terminal electron acceptor. However, all strains can use nitrate as an alternative electron acceptor and can carry out oxygen-repressible denitrification. Denitrification may be delayed or may appear only after serial transfers in nitrate media under semiaerobic conditions (73, 340). Oxidative degradation of aromatic compounds involves the participation of mono- and dioxygenases. Typically, catechol or protocatechuate is the central intermediate in this reaction. Each is cleaved through an ortho pathway when no accessory genes are involved in the degradation. Amylolytic activity is one of the phenotypic characteristics of the species. The enzymology of the exo-amylase—which is responsible for the formation of maltotetraose as an end product—has been examined at the molecular level. This enzyme has also been cloned (231). Obradors and Aguilar demonstrated that polyethylene glycol was degraded to yield ethylene glycol, a substrate typically used by P. stutzeri strains (241).
The arginine deiminase system ("dihydrolase") catalyzes the conversion of arginine to citrulline and of citrulline to ornithine. It has been used by taxonomists to differentiate species. All P. stutzeri strains give a negative test result for this reaction. They also fail to use glycogen and do not liquefy gelatin.
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Caution should be exercised when only phenotypic traits are used for classification. This can clearly be seen in the case of strain ZoBell. This strain (ATCC 14405) was isolated as a marine bacterium and described by ZoBell and Upham as "Pseudomonas perfectomarinus" in 1944 (412). Subsequently, this organism became the only member of the species P. perfectomarina. Its lack of flagella was emphasized by its assignation to a new species, although the authors who first described this strain stated that it was motile (19, 412). After three passages, enrichment for flagellated bacteria on semisolid tryptone agar enabled a population in which over 80% of cells were flagellated to develop. This revertant strain is motile by means of a single polar flagellum (294).
In a recently published chapter on chemotaxis in Pseudomonas, Parales et al. stated, "All Pseudomonas species are motile by one or more polar flagella and are highly chemotactic" (258). P. stutzeri is no exception. Chemotaxis machinery has not been studied in detail for any Pseudomonas species. Moreover, the ranges of attractants or repellents and environmental conditions to which Pseudomonas spp. respond remain largely unexplored. They seem to be attracted to virtually all of the organic compounds they can use as growth substrates. However, they are also attracted to other compounds that they are unable to metabolize. Ortega-Calvo et al. studied the chemotactic response of several pseudomonads to polycyclic aromatic hydrocarbon-degrading bacteria (243). Strain 9A of P. stutzeri was included in the study. This strain degrades naphthalene, phenanthrene, and anthracene. It was concluded that chemotaxis was positive to naphthalene and to the root exudates of several plants. Chemotaxis may enhance the biodegradation of pollutants in the rhizosphere, at least in laboratory-scale microcosms. Strain KC mineralizes carbon tetrachloride, and motility-enhanced bioremediation in aquifer sediments has been demonstrated (401, 402).
Pseudomonas species have a range of different adhesins that function during initial attachment to a substratum. This leads to biofilm formation. Both flagella and pili seem to be important in the colonization of biotic and abiotic surfaces, particularly in the initial formation of microcolonies. P. aeruginosa's initial biofilm development appears to be conditionally dependent on type IV pili. P. stutzeri possesses both flagella and pili but has not been described as a member of consortia that form natural biofilms. Type IV pili confer twitching motility to P. stutzeri strains (a bacterial movement based on pilus extension/retraction). This is probably at least partly responsible for many colonies' diffuse borders (J. Sikorski, personal communication). These colonies also correspond to strains that have natural transformation ability.
Protein patterns. Whole-cell protein patterns obtained by denaturing polyacrylamide gel electrophoresis (PAGE) are highly characteristic at the strain level. They have been used for typing and classification purposes (265). P. stutzeri strains have been found to be particularly heterogeneous (271, 295). Computer-assisted analysis of the protein bands creates a dendrogram that is in good agreement with the genomovar subdivision of the species (366). This result is not surprising, as whole-cell protein patterns reflect the protein-encoding genes in the whole genome and the genomovars were defined by the similarity values of total DNA-DNA hybridizations.
LPS and immunological characteristics. Lipopolysaccharide (LPS) is the main antigenic molecule on the cell surface. This is considered to be the heat-stable O-antigen of the genus. The specificity of antibodies is related to the composition of the polysaccharide chains projecting outside the cells. Representative P. stutzeri strains of the seven known genomovars on which experiments were done showed marked serological diversity. This parallels the LPS O side-chain heterogeneity between strains. In the study by Rosselló et al., antigenic relatedness was observed only between closely related strains of the same genomovar (292).
Outer membrane proteins analyzed by sodium dodecyl sulfate-PAGE gave very similar results for all strains tested, regardless of genomovar ascription. Likewise, similar results were attained for immunoblotting using polyclonal antisera against six representative strains' whole cells. However, a similar procedure, based on Western blotting and immunological fingerprinting of whole-cell proteins using the polyclonal antibody Ab160, raised against Pseudomonas fluorescens MT5—called Westprinting (360)—produced a typical protein profile for each strain. Computer-assisted comparisons revealed a distribution in groups that agreed with the strains' genomovar distribution at different similarity levels (25).
Fatty acid composition. Fatty acid composition is a very good taxonomic marker for distinguishing the genus from other genera formerly included in Pseudomonas (e.g., Burkholderia). These chemotaxonomic characteristics are very useful for identification purposes. Studies of the fatty acid composition of Pseudomonas species (158, 246, 341, 367) revealed that the straight-chain saturated fatty acid C16:0 and the straight-chain unsaturated fatty acids C16:1 and C18:1 were the most abundant. These account for 82.3% of total fatty acids in P. stutzeri. Minor quantities of the hydroxylated fatty acids 3-OH 10:0 and 3-OH 12:0 were also detected (295). There were no significant differences between genomovars in the other fatty acids. Members of genomovar 6 had a higher content of cis-9,10-methylenehexadecanoate (17:0) and cis-9,10-methyleneoctadecanoate (19:0). This chemotaxonomic particularity, together with other characteristics, helped to distinguish genomovar 6 as a new species, Pseudomonas balearica (23).
Fatty acid composition must be determined under strictly controlled growth conditions, as it is highly dependent on growth substrates. Mrozik et al. describe the changes in fatty acid composition in strains of P. putida and P. stutzeri during naphthalene degradation (232, 233). The reaction of both strains to the addition of naphthalene was an increase in the saturated/unsaturated ratio and alterations in the percentage of hydroxy, cyclopropane, and branched fatty acids. New fatty acids were detected when the strains were exposed to naphthalene.
Quinone and polyamine composition. The determination of polyamine and quinone composition is a rapid chemotaxonomic identification tool. Putrescine is the main component of all members of the genus Pseudomonas (57). Two major polyamines were detected in P. stutzeri: putrescine (35.0 to 92.7 µmol/g [dry weight]) and spermidine (8.9 to 29.2 µmol/g [dry weight]). Other polyamines were detected in very small amounts only (1,3-diaminopropane, cadaverine, and spermine) (293). Ubiquinone Q-9 is the only quinone present in all of the P. stutzeri strains studied.
PHA. P. stutzeri cells do not accumulate polybetahydroxybutyrate. However, the production of novel polyhydroxyalkanoates (PHA) by one strain of the species (strain 1317) has been demonstrated (141). This strain was isolated from oil-contaminated soil in an oil field in northern China. Another P. stutzeri strain, YM1006, has been isolated from seawater as a poly(3-hydroxybutyrate)-degrading bacterium, although it does not seem to be able to accumulate this reserve material. The extracellular polybetahydroxybutyrate depolymerase gene (phaZPst) has been well characterized (242).
Some combinations of unusual phenotypic properties can be very helpful in the preliminary assignment of newly isolated strains to certain species. Alternatively, the absence of one or more of the set's properties suggests that the strain should be excluded from the taxon. For example, in addition to the basic characteristics of a Pseudomonas species, the following characteristics strongly suggest that a culture is a strain of Pseudomonas stutzeri: denitrification with copious gas emission; the formation of dark, folded, coherent colonies; and the capacity to grow at the expense of starch, maltose, or ethylene glycol. However, in our laboratories we have found that enrichment conditions frequently yield cultures lacking one or more of the key characteristics mentioned above. Such enrichment conditions included the use of aromatic compounds and some of their halogenated derivatives as the sole carbon and energy sources. Although the general phenotypic properties of these cultures could be used a priori as an argument for excluding them from the species, it was surprising to find that some of them were phylogenetically very similar to P. stutzeri. This is probably true in the case of a strain ascribed to Pseudomonas putida in a patent for the mineralization of halogenated aromatic compounds (U.S. patent no. 4,803,166, 7 February 1989). Its DNA sequences most probably indicate its affiliation to P. stutzeri. Detailed analysis of atypical phenotypes (such as the absence of either motility or denitrification) demonstrated in some cases that the characteristic was cryptic and could be expressed when the cells were adapted.
An interesting example of variation to be taken into consideration may be the lack of folded colonies, which, in principle, is taken as an important primary criterion for the isolation. In fact, the discovery of P. mendocina at the University of Cuyo, Mendoza, Argentina, was linked to isolations of smooth colonies of Pseudomonas which at first were taken to be biovars of P. stutzeri.
| GENOMIC CHARACTERIZATION AND PHYLOGENY |
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Tm] values). Members of two different P. stutzeri genomovars have 15 to 50% DNA-DNA similarity values or Tm value differences greater than 5°C. Subsequently, this concept has been used taxonomically to group genotypically similar strains in other species, such as Burkholderia cepacia and species in the genera Xanthobacter, Azoarcus, and Shewanella, etc. It provides a useful provisional level of classification.
The methods used to calculate DNA-DNA similarity values have differed from one laboratory to another. Palleroni used 125I labeling and/or membrane filters (251). Rosselló et al. used the Tm method, as described previously (291). Sikorski et al. used the method described by Ziemke et al. (411), with digoxigenin and biotin labeling and quantification of the binding ratio in microtiter plates (327). Vermeiren et al. used DNA-DNA thermal reassociation, measured photometrically (380). The results were consistent with the genomovar subdivision of the species, regardless of the method used to estimate the similarity value.
To date, nine different genomovars have been well documented. Eight new genomovars in the species P. stutzeri were put forward recently (327). One reference strain has been proposed for each genomovar and deposited in culture collections. Most strains studied so far are included in genomovar 1 (along with the species' type strain). The genomovars 8 (strain JM300), 9 (strain KC), 10 (strain CLN100), and 18 (strain MT-1) each have only one representative strain. These might be considered genomospecies, sensu Brenner et al. (50). As an example, we can consider strain CLN100, of genomovar 10. It is a representative of a new species from a genomic perspective, sharing many substantial phenotypic and phylogenetic characteristics with members of the P. stutzeri phylogenetic branch. Some phenotypic traits can be used to discriminate CLN100 from the P. stutzeri and P. balearica strains described to date (simultaneous degradation of chloro- and methyl-derivatives of naphthalene and absence of ortho cleavage of catechol, etc.). These characteristics could be the basis for describing CLN100 as the type strain of a new species. However, some of these phenotypic traits could be strain specific; therefore, it was preferred not to define a new species until more strains that are genomically and phenotypically similar to strain CLN100 have been described (114).
From one to four plasmids were detected in 10 of the 20 strains analyzed in this study (121). The Eckhardt method, using both conventional and pulsed-field gel electrophoresis, turned out to be the most reliable and useful technique for plasmid detection. Seventy-two percent of the plasmids observed were smaller than 50 kb, one plasmid was between 50 and 95 kb, and four plasmids were larger than 95 kb. No two strains shared the same plasmid profile, and no relation was found between genomovars and the distribution of plasmids among the strains. Seven of the 10 plasmid-containing strains were isolated from polluted environments. This is not uncommon in plasmid analyses. A correlation between the degree of contamination and the incidence of plasmid occurrence was found in an environmental study by Baya et al. (20). Naphthalene degradation plasmids are common in Pseudomonas spp. However, in eight of the nine naphthalene-degrading strains of P. stutzeri studied, the catabolic genes were inserted into an I-CeuI chromosomal fragment, as demonstrated by Southern blot hybridizations with nahA and nahH probes. The naphthalene genes seem to be plasmid encoded only in strain 19SMN4 (120, 296).
Methods based on the electrophoretic patterns of macrorestriction fragments (low-frequency restriction fragment analysis) have been used by two independent groups to examine representative strains (121, 271). The restriction enzymes XbaI and SpeI cut the P. stutzeri genome of the strains studied into 20 to 48 fragments. These fragments were resolved by pulsed-field gel electrophoresis. They are useful for generating fingerprints, which can be used to explore genome structures and to determine the degree of relatedness of strains. No correlation was found between the similarity of macrorestriction patterns and the subdivision of the species into genomovars. This was due to the high discriminatory power of the two enzymes and the heterogeneity of the restriction patterns. However, some patterns allowed clonal variants between strains to be distinguished. In these cases the related strains belonged to the same genomovar. The marked heterogeneity was attributed, at least in part, to large chromosomal rearrangements (121).
In the ribotyping procedure, total DNA is purified and then cleaved by restriction endonucleases. Brosch et al. (51) used the enzymes SmaI and HincII in their study of Pseudomonas strains. Restriction fragments were separated by electrophoresis, transferred to a nylon membrane, and hybridized with a 16S-23S rRNA probe. Nine strains of P. stutzeri clustered together in the dendrogram, which also showed 217 other strains from different Pseudomonas species. Two identical bands were detected by HincII in P. stutzeri. SmaI profiles were more discriminative, distinguishing from four to eight bands. Members of a single genomovar were grouped in the same branch.
Bennasar et al. (25) revealed genetic diversity and the relationships among P. stutzeri strains by rapid molecular typing methods. Repetitive extragenic palindromic PCR and enterobacterial repetitive intergenic consensus PCR analyses, based on DNA consensus sequences, generated fingerprints that were then computer analyzed. Groupings were consistent with the genomic groups that had previously been established by DNA-DNA hybridizations or 16S rRNA sequencing. Members of other Pseudomonas species were clearly different. Sikorski et al. (325) carried out random amplified polymorphic DNA (RAPD) PCR analysis in their study of P. stutzeri isolates from marine sediments and soils in geographically restricted areas (local populations). The results demonstrated the complex composition and high strain diversity of the local populations studied.
Similar genomic relationships have been revealed by PCR amplification of several genes (16S rRNA, internal transcribed spacer region 1 [ITS1], ITS2, and rpoB) and by analyzing the RFLPs generated by several restriction enzymes (25, 133, 325). These methods have confirmed the high genetic diversity of the species, the consistency of genomic groups (genomovars), and the usefulness of the patterns generated for strain identification.
Two independent research groups have used the MLEE approach in studies of P. stutzeri (284, 324). In Sikorski's study, 16 P. stutzeri strains belonging to eight different genomovars were analyzed for the allelic profiles of 21 enzymes. A distinctive multilocus genotype was detected in all strains, and up to 11 alleles were detected per locus. In Rius's analysis, 42 P. stutzeri strains from nine genomovars (including 9 strains previously studied by Sikorski et al.) and 20 enzymes were studied. The highest number of different alleles found per locus was 32, and all multilocus genotypes were represented by a single strain. Forty-two electrophoretic types were detected. In both analyses, P. stutzeri was shown to have a highly polymorphic structure. If both groups' results are combined, 49 different P. stutzeri strains have been studied with MLEE. A total of 33 different enzymes were analyzed from these strains. An analysis of this set of 49 strains again demonstrates that all of the multilocus genotypes were represented by a single strain. MLEE studies reveal that P. stutzeri is highly polymorphic. The highest genetic diversity described for a species is revealed (284) by an analysis of the members of genomovar 1 only. An analysis of source and place of isolation showed no clear association in clusters. When two subgroups of P. stutzeri populations (clinical and environmental isolates) were compared, the mean levels of genetic diversity were not significantly different. This indicates that clinical strains come from the same populations as environmental isolates. This may have important epidemiological implications for the microbiology of P. stutzeri infections. However, when two strains were grouped at moderate genetic distances (below 0.55), each pair of strains belonged to the same genomovar.
All loci were highly polymorphic in the 26 strains studied. The number of nucleotide substitutions per nucleotide site varied from 44.2% for catA to 21.8% for nahH. The number of alleles varied in the different loci: 4 in nahH (16 strains), 18 in catA (24 strains), 20 in gyrB (26 strains), 17 in rpoD (26 strains), 18 in nosZ (26 strains), 15 in 16S rRNA (26 strains), and 20 in ITS1 (26 strains). Apart from nahH (a gene that is probably acquired through lateral transfer), the mean number of alleles per locus in the 26 strains was 18.7, an extremely high value. The average number of alleles per locus and strain was 0.72.
In this MLST study (72), the dN/dS ratio—the ratio of nonsynonymous substitutions per nonsynonymous site which resulted in an amino acid replacement (dN) to synonymous substitutions per synonymous site that did not change the amino acid (dS)—was calculated for the genes encoding proteins as a measure of the degree (amount and type) of selection in P. stutzeri populations. Changes are selectively neutral when they are independent of the overlying phenotype and the selection pressure dictated by the phenotype's function. The ratio was less than 0.1 in three genes (gyrB, rpoD, and nosZ). The highest dN/dS ratio corresponded to catA (0.18). All ratios were much less than 1, indicating that these gene fragments are not under selection. In other words, most of the sequence variability identified is selectively neutral. Synonymous substitutions were at least 5.5 times (1/0.18) more frequent than amino acid changes at any locus.
The number of nucleotide substitutions per nucleotide site was higher than in Campylobacter jejuni, Neisseria meningitidis, Streptococcus pneumoniae, Enterobacter faecium, and species of the Bacillus cereus complex. To our knowledge, the number of nucleotide substitutions described for P. stutzeri is the highest recorded to date (145). The average numbers of alleles per locus and strain analyzed in the protein-coding genes were 0.72 for P. stutzeri (an average of 18.7 alleles per locus in only 26 strains), 0.18 for C. jejuni, and 0.43 for the B. cereus complex. These values are in good agreement with previous observations made in MLEE studies of most of the strains analyzed by the MLST technique. In such MLEE studies the genetic diversity was the highest described for a species (284). Therefore, the extremely high genetic diversity of the species manifested by MLEE was corroborated by the MLST study.
Figure 2 shows an analysis of the sequence types (STs) identified among 26 independent strains of P. stutzeri. This analysis led to the assumption that one different ST per strain can be detected. This is the highest possible number of STs. Remarkably, when two strains had an allele in common they belonged to the same genomovar. There was only one exception: strain JM300 (genomovar 8) has an rpoD allele that is identical to strain JD4, one of the two members of genomovar 5. This can be explained by genomovars 5 and 8 having a common ancestor or by a possible lateral gene transfer to JM300, a strain intensively studied due to its natural transformation (206). Another strain, AN10 of genomovar 3, presents a possible recombination event with members of the same genomovar. Strains 19SMN4 and ST27MN3, of genomovar 4, were very closely related in the multilocus sequence analysis. They had identical 16S rRNA, rpoD, and gyrB genes. Both strains were isolated as naphthalene degraders from samples taken in a wastewater treatment lagoon. However, they were from different habitats (water column and sediment). Molecular typing methods (25, 121, 133) and MLEE (284) had previously demonstrated that both strains were genetically related but different. Again, the enormous genetic diversity of the species was demonstrated in this study. Inclusion of nahH in the analysis modifies the topography of the graph, indicating more possible events of lateral gene transfer (Fig. 2).
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Phylogenetic tree reconstructions of the same genes used in the MLST method (16S rRNA, ITS1, gyrB, rpoD, nosZ, and catA) were undertaken by Cladera et al. (72). Stability analysis using bootstrap resampling showed that the trees were stable and well defined. Most strains of P. stutzeri clustered in the same phylogenetic branch in the gene trees analyzed. They were usually separated from the other closely related species considered, P. balearica and P. mendocina. Strains belonging to the same genomovar were usually located in the same branch. There were only a few exceptions, which varied depending on the gene analyzed. A consensus phylogenetic tree was constructed for the six genes to deduce a composite molecular phylogeny for P. stutzeri. All P. stutzeri strains are located in the same phylogenetic branch, and members of each genomovar are clustered together, maintaining the genomovar subdivision of the species. This tree is based on a sequence of no less than 4,551 nucleotides, representing at least 9,546 nucleotides from the respective genomes, as there are four copies of the rrn operon in P. stutzeri. Therefore, between 0.2 and 0.25% of the chromosome (depending on the strain's genome size) has been compared pairwise in 24 independent isolates.
A careful analysis of some genes, based on incongruences in the phylogenetic trees and/or what is known as relative codon usage, the codon bias index, or the G+C content of the genes, can help to define some metabolic pathways as genes acquired through horizontal transfer. The following examples are considered below: the aromatic degradative pathway, the nitrogenase system, the ability to use chlorate as a terminal electron acceptor, and the energy-yielding reactions in the oxidation of thiosulfate.
| TAXONOMIC RANKS: GENOMOVARS |
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Subspecies designations can be used for organisms that are genetically close but phenotypically divergent. In this way, the infraspecific level seems to be phylogenetically valid. It can be distinguished from the infrasubspecific concept of variety. This concept is based solely on selected "utility" attributes that cannot be demonstrated by DNA reassociation (392). Ranks below subspecies are often used to indicate groups of strains that can be distinguished by some special characteristic. Such ranks have no official standing in nomenclature but often have great practical usefulness. An infrasubspecific taxon is one strain or a set of strains that have the same or similar properties and are treated as a taxonomic group.
The "genomovar" concept was coined (291, 363) to clarify the taxonomic status of P. stutzeri genomic subgroups. Therefore, the concept was first applied to P. stutzeri. It is a useful pragmatic approach to classifying individual strains when they are genomically different from phenotypically closely related strains. It is also of use when phenotypic intragroup variability cannot be clearly established. This occurs when only a small set of strains (or just one) has been isolated. There is no clear phenotypic or biochemical relationship, or a common geographical origin or source of isolation, between members of the same genomovar in P. stutzeri. The suffix "-var" refers to a taxonomic rank below the species level. Nine genomovars (114) have been intensively studied within the species. Members of two different genomovars are genomically distant enough to be considered different genomic species. However, due to the lack of discriminative phenotypic traits, the strains are included in the same nomenspecies. Recent studies undertaken by Sikorski et al. (327) and Romanenko et al. (289) have described some additional P. stutzeri isolates that belong to previously described genomovars and others that represent at least eight new genomovars. These results were obtained by 16S rRNA phylogenetic analysis, RAPDs, and DNA-DNA hybridizations (327).
Since its definition, the genomovar concept has been applied to other genomic groups in different bacterial species, such Burkholderia cepacia (368) and Azoarcus spp. (336). It could be applied to other well-defined genomic groups in species such as Shewanella putrefaciens and Bacillus cereus, etc. Other authors (e.g., J. P. Euzéby [http://www.bacterio.cict.fr/]) consider "genomovar" to be an unfortunate term, as it assumes that genomic differentiation should be the basis for differentiating bacterial species.
Due to the high genomic diversity of P. stutzeri strains, other authors prefer to use supraspecific terms to refer to all of them. Examples are the P. stutzeri "group" (337), the P. stutzeri "superspecies" (337), and the P. stutzeri "complex" (408).
| IDENTIFICATION |
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A microbial cell expresses some 200 different proteins that can be separated by PAGE. This yields complex banding patterns, which are considered to be highly specific fingerprints (265). Strains with at least 70% DNA similarity tend to have similarities in protein electrophoretograms. Therefore, PAGE is thought to be a sensitive technique for gaining information on the similarities between strains within the same species or subspecies. Individual strains can often be recognized by protein pattern. Under standard growth and PAGE conditions, the patterns are reproducible. Computer-assisted analysis enables the information to be normalized and stored. This method has been used to identify P. stutzeri strains when a wide database is available (366).
The Sherlock microbial identification system is based on analyzing total fatty acid profiles. It gives satisfactory results within the genus Pseudomonas, including P. stutzeri, if the cells are cultured under strictly controlled conditions.
A second set of primers, fps158 and rps1271, was developed by Bennasar et al. (24). These primers produced a 1,159-bp amplicon containing a BamHI restriction site. The specificity of the amplicon for P. stutzeri was then corroborated by restriction, giving two fragments, of 695 and 465 bp, respectively. A slightly modified set of primers in the same region was used successfully by Sikorski et al. (325).
The three methods permit good molecular differentiation of P. stutzeri from other species. They have been used to identify P. stutzeri and to detect it in environmental samples, as indicated below (see "Occurrence and Isolation Procedures").
| PHYSIOLOGICAL PROPERTIES |
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Some strains (NF13, MT-1) have been isolated from the deep-sea bottom. Organisms adapted to the deep-sea environment have to grow under conditions of 2°C and 100-MPa pressure. On 28 February 1996, a sediment sample was obtained from the Mariana Trench by the unmanned submersible Kaiko. It seems likely that this was the first time sediment samples were collected from the world's deepest point without any microbiological contamination from other depths (351). The analysis of amplified 16S rRNA sequences from DNA directly extracted from these sediment samples demonstrated the presence of bacteria belonging to the P. aeruginosa branch (Mariana bacteria no. 2 [D87347] and no. 11 [D87346]). Pressure-regulated gene clusters were also amplified. Therefore, in addition to being barotolerant, the bacteria from the Mariana sediment may be barophilic microorganisms. Barophilic microorganisms were isolated by maintaining the conditions of 100 MPa and 4°C. Twenty-eight strains were selected. Strain MT-1, isolate HTA208, was grown on marine agar at 28°C and pH 7.6. Its 16S RNA sequence affiliates the strain with the P. stutzeri phylogenetic branch. It was able to grow at a hydrostatic pressure of 30 to 60 MPa, and slight growth occurred at 100 MPa. The growth rate of the P. stutzeri type strain was strongly affected by hydrostatic pressure. It must be clarified whether the isolated bacteria are active or inactive under high hydrostatic pressure and low temperature or whether their presence is simply a result of settling of flocculated organic matter.
As mentioned above, no strain tolerates acidic conditions: all fail to grow at pH 4.5. This is probably the reason why there is a negative reaction to the oxidation/fermentation test for the use of carbohydrates. Many P. stutzeri strains give a neutral result, as the medium is not buffered and acidification inhibits further growth, even when the strain might be able to use the added sugar.
P. stutzeri strains grow well under atmospheric oxygen. However, microaerophilic conditions have to be established when nitrogen-fixing strains are cultured as diazotrophs. All strains described to date are facultatively anaerobic with nitrate. Some strains are also anaerobic, with chlorate or perchlorate as terminal electron acceptors. Both anaerobic properties are discussed in the following section.
In contrast to the assimilatory reduction of nitrate or nitrite to ammonia for biosynthetic purposes, denitrification in bacteria is a dissimilatory transformation, associated with energy conservation (420). In other words, the enzymatic electron transfer is coupled to ATP synthesis via proton translocation and the formation of a membrane potential (347). The bacterial process of denitrification is normally a facultative trait. It provides bacteria with a respiratory pathway for anaerobic life (184, 420). The distribution of denitrification capabilities among the prokaryotes does not follow a clear pattern (263). The former genus Pseudomonas is one of the largest taxonomic clusters of known denitrifying<
