Pseudomonas Genomes: Diverse and Adaptable Mark W

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REVIEW ARTICLE Pseudomonas genomes: diverse and adaptable Mark W. Silby1,2, Craig Winstanley3, Scott A.C. Godfrey4, Stuart B. Levy2 & Robert W. Jackson5

1Department of Biology, University of Massachusetts Dartmouth, North Dartmouth, MA, USA; 2Department of Molecular Biology and Microbiology, Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, Boston, MA, USA; 3Institute of Infection and Global Health, University of Liverpool, Liverpool, UK; 4Centre for Research in Plant Science, University of the West of England, Bristol, UK; and 5School of Biological Sciences, The University of Reading, Reading, UK Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021

Correspondence: Mark W. Silby, Abstract Department of Biology, University of Massachusetts Dartmouth, 285 Old Westport Members of the genus Pseudomonas inhabit a wide variety of environments, which Road, North Dartmouth, MA 02747, USA. is reflected in their versatile metabolic capacity and broad potential for adaptation Tel.: 11 508 999 8364; fax: 11 508 999 to fluctuating environmental conditions. Here, we examine and compare the 8196; e-mail: [email protected] genomes of a range of Pseudomonas spp. encompassing plant, insect and human pathogens, and environmental saprophytes. In addition to a large number of allelic Received 22 May 2010; revised 26 January differences of common genes that confer regulatory and metabolic flexibility, 2011; accepted 15 February 2011. genome analysis suggests that many other factors contribute to the diversity and Final version published online 25 March 2011. adaptability of Pseudomonas spp. Horizontal gene transfer has impacted the capability of pathogenic Pseudomonas spp. in terms of disease severity (Pseudomo- DOI:10.1111/j.1574-6976.2011.00269.x nas aeruginosa) and specificity (Pseudomonas syringae). Genome rearrangements

Editor:Miguel Camara likely contribute to adaptation, and a considerable complement of unique genes undoubtedly contributes to strain- and species-specific activities by as yet Keywords unknown mechanisms. Because of the lack of conserved phenotypic differences, adaptability; bacterial species concept; the classification of the genus has long been contentious. DNA hybridization and comparative genomics; Pseudomonas genome. genome-based analyses show close relationships among members of P. aeruginosa, but that isolates within the Pseudomonas fluorescens and P. syringae species are less closely related and may constitute different species. Collectively, genome sequences of Pseudomonas spp. have provided insights into pathogenesis and the genetic basis for diversity and adaptation.

aided taxonomic definition and species reorganization (Pal- Introduction leroni & Moore, 2004). Based largely on data from molecular Members of the genus Pseudomonas (sensu stricto) show studies, strains thought to belong to Pseudomonas sensu remarkable metabolic and physiologic versatility, enabling stricto (in the Gamma-subclass of Proteobacteria) have been colonization of diverse terrestrial and aquatic habitats separated from the genus and placed into the genera (Palleroni, 1992), and are of great interest because of their Burkholderia, Ralstonia and Comamonas (Betaproteobacteria) importance in plant and human disease, and their growing (Kersters et al., 1996). Whole-genome sequences provide an potential in biotechnological applications (Fig. 1). Since the opportunity to further address the systematics of the genus. genus Pseudomonas was first described, the assignment of Pseudomonas aeruginosa PAO1 was the 25th bacterial isolates to and within the genus has been contentious. genome sequence to be completed (Stover et al., 2000). Stanier et al. (1966) published a comprehensive appraisal of Genome sequences of strains of Pseudomonas syringae (Buell the taxonomy of Pseudomonas spp., largely determined by et al., 2003), Pseudomonas putida (Nelson et al., 2002), phenotypes and biochemical capabilities. The resolution of Pseudomonas fluorescens (Paulsen et al., 2005), Pseudomonas its intrageneric structure using DNA–DNA hybridization entomophila (Vodovar et al., 2006), Pseudomonas mendocina

MICROBIOLOGY REVIEWS MICROBIOLOGY (DDH), analysis of rRNA and housekeeping gene sequences (unpublished data) and Pseudomonas stutzeri (Yan et al., and multilocus sequencing (Palleroni et al., 1973; Moore 2008) followed, and have since been augmented by addi- et al., 1996; Maiden et al., 1998; Gardan et al., 1999; Anzai tional sequences of several strains of these bacterial species. et al., 2000; Yamamoto et al., 2000; Goris et al., 2007) have The genome of the nitrogen-ﬁxing bacterium Azotobacter

Fig. 1. The functional and environmental range of Pseudomonas spp. The Pseudomonas common ancestor has encountered a wide range of abiotic and biotic environments that has led to the evolution of a multitude of traits and lifestyles with significant overlap among species. vinelandii, classified in the Pseudomonadaceae, has also been studies of systematics and evolution. We shall not specifi- sequenced (Setubal et al., 2009). In each case, genome cally review the genomics of secondary metabolite produc- sequencing was undertaken to identify the genetic features tion. Instead, we direct readers to an excellent review by contributing to the particular lifestyle or phenotype of Gross & Loper (2009). interest for the sequenced isolate. As of January 2011, there were 18 complete Pseudomonas genomes listed in NCBI’s Genome sequence features Entrez database, with another 72 listed as being draft assemblies or incomplete. Of these 90, 15 are P. aeruginosa ‘Core’ and ‘accessory’ genomes and the isolates (strain PA14 is listed in both complete and draft conserved gene set project pages) and 38 are pathovars of P. syringae (strain B728a is listed in both complete and draft project pages). The concept of a ‘core’ genome has been used to describe the That close to 65% of sequences are derived from just two conserved sequences in P. aeruginosa, with the remaining pathogenic members of the genus demonstrates the con- genes comprising the accessory genome, which has been siderable importance of these Pseudomonas spp. in human defined as the set of genes missing from one or more strain and plant health. (Mathee et al., 2008). Because the core group of genes is Here, we review the genomes of Pseudomonas spp., first refined each time a new genome sequence is released, the considering the genome structures of the bacteria. These are core gene complement is defined as the conserved set of considered in the context of the lifestyle and properties of genes common to a particular group of organisms under Pseudomonas spp. with an emphasis on diversity and adapt- consideration. This core set will reduce in size as additional ability. We consider the potential uses of genomics for genome sequences are determined, and it is not possible to

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 654 M.W. Silby et al. know how many sequences will be required before a as rhizosphere colonizers (Haas & Defago, 2005). Major ‘plateau’ is reached where the true core gene complement secondary metabolites produced by Pseudomonas strains can conﬁdently be assigned. Ignoring the large inversions have been detected and investigated because of their anti- found in some P. aeruginosa strains, the regions of conserved microbial activity (Leisinger & Margraff, 1979) and include genes could be mapped onto the PA14 genome as a 2,4-diacetylphloroglucinol (Raaijmakers et al., 1997), template, revealing the location and size of strain-speciﬁc pyoluteorin (Howell & Stipanovic, 1980), pyrrolnitrin segments, many of which originate from horizontal gene (Howell & Stipanovic, 1979), hydrogen cyanide (Voisard transfer (Mathee et al., 2008). et al., 1989), syringomycin (Sorensen et al., 1996), syringopeptin (Lavermicocca et al., 1997), viscosinamide (Nielsen Genomic islands (GIs), phage and plasmids -- the et al., 1999), viscosin (Neu et al., 1990; de Bruijn et al.,

accessory genome in Pseudomonas spp. 2007), thioquinolobactin (Matthijs et al., 2007), phenazines Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 (Thomashow & Weller, 1988) and yet to be identified Mobile genetic elements (MGEs) such as phages, plasmids, compounds (Garbeva & de Boer, 2009). Pseudomonads transposons and GIs are frequently identified in microbial are also able to produce insecticides, including P. fluorescens genomes, and can play significant roles in processes such as Pf-5 Fit (for P. fluorescens insecticidal toxin) against tobacco pathogenesis and antibiotic resistance (Arnold et al., 2003; hornworm (Pechy-Tarr´ et al., 2008); P. entomophila with Hacker et al., 2004; Jackson et al., 2011). In Pseudomonas currently unknown toxin(s) against Drosophila (Vodovar spp., MGEs have been identified in all species in which they et al., 2005); and P. syringae with an unknown action against have been sought. While these often follow the established aphids (Stavrinides et al., 2009). patterns (e.g. integration into tRNA genes), there is con- Bioremediation uses microorganisms to degrade or de- siderable variability in the identity, location and functional toxify hazardous environmental contaminants. The excep- gene content, even within strains of a given species. tional nutritional versatility of pseudomonads, coupled with Most species have at least one prophage-like element (some the production of biosurfactants that can mobilize hydro- may be functional while others are degraded remnants), carbons and nonaqueous phase liquids into an aqueous several coding sequences (CDSs) with similarity to transpo- phase (Desai & Banat, 1997), makes them excellent sase genes and plasmid-related sequences. The influence of candidates for bioremediation. Pseudomonas aeruginosa is MGEs on Pseudomonas genomes is considerable in some frequently isolated in petroleum-contaminated soils and cases, but appears to be less prominent in P. entomophila groundwater (Ridgway et al., 1990). Pseudomonas putida (Vodovar et al., 2006) and P. stutzeri (Yan et al., 2008), has been extensively studied in environmental biotechnol- although key nitrogen-fixation functions in the latter are ogy because of its capabilities in the bioremediation of toxic encoded on a GI. The accessory genome will be described in organic wastes including aromatic hydrocarbon compounds more detail later in this review. (Loh & Cao, 2008). Other Pseudomonas species identified with bioremediation properties include P. mendocina Repetitive extragenic palindromic (REP) (Whited & Gibson, 1991) and P. stutzeri (Grimberg, 1996). elements Several studies have used screening technologies to iden- These DNA sequences vary in length between 21 and 65 bp tify genes important for soil, and plant root (rhizosphere) and are found in extragenic regions of the genomes of some and leaf (phyllosphere) colonization (Rainey, 1999b; Boch bacteria (Tobes & Pareja, 2006). Their role is not precisely et al., 2002; Gal et al., 2003; Rediers et al., 2003; Silby & Levy, defined, but because they can stabilize mRNA and are the 2004; Marco et al., 2005; Ramos-Gonzalez et al., 2005). target for DNA polymerase I, DNA gyrase, integration host However, most of these screens were limited in scope factor and transposases and recombinases, they may play a because many of the genetic screens were proof of principle significant role in genome evolution and adaptation (re- and did not screen the genome to saturation, thus only viewed in Tobes & Pareja, 2006). REP sequences are dis- providing limited genome coverage. A major driver for cussed throughout this review. sequencing the genomes of these bacteria was to gain a deeper insight into bacterial function, both in terms of their Plant growth promotion and niche colonization and their metabolic and physiological properties. bioremediation Many pseudomonads interact with plants and several spe- Pseudomonas putida KT2440 -- a cies contribute to plant health by antagonizing plant-patho- biodegradation bacterium genic microorganisms (biocontrol) and directly influencing plant disease resistance and growth (plant growth promo- Pseudomonas putida are soil- and plant-associated bacteria tion) – both as plant endophytes (Ryan et al., 2008) and of interest for their biodegradation properties. Like most

Table 1. General features from completed Pseudomonas genomes Species/strain SizeÃ %G1CÃ Genesw % coding tRNAw rRNAw Released GenBank Reference P. aeruginosa PAO1 6 264 404 66.6 5671 89.8 63 13 13/09/2000 AE004091.2 Stover et al. (2000) PA7 6 588 339 66.4 6396 90.1 65 12 05/07/2007 CP000744.1 Roy et al. (2010) UCBPP-PA14 6 537 648 66.3 5994 89.8 63 13 06/10/2006 CP000438.1 Lee et al. (2006) LESB58 6 601 757 66.3 6026 88.9 67 13 24/12/2008 FM209186.1 Winstanley et al. (2009) C3719 6 222 097 66.5 5696 86.6 40 6 04/01/2006 NZ_AAKV00000000z Mathee et al. (2008) PA2192 6 905 121 66.2 6203 85.5 44 2 04/01/2006 NZ_AAKW00000000z Mathee et al. (2008) P. entomophila L48 5 888 780 64.2 5293 89.8 78 22 10/05/2006 CT573326.1 Vodovar et al. (2006)

P. ﬂuorescens Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 Pf0-1 6 438 405 60.6 5741 90.0 73 19 07/10/2005 CP000094.2 Silby et al. (2009) Pf-5 7 074 893 63.3 6144 88.7 71 16 30/06/2005 CP000076.1 Paulsen et al. (2005) SBW25 6 722 539 60.5 6009 88.3 66 16 09/01/2008 AM181176.4 Silby et al. (2009) P. mendocina ymp 5 072 807 64.7 4730 90.7 67 12 20/04/2007 CP000680.1 Unpublished P. putida F1 5 959 964 61.9 5423 89.9 76 20 31/05/2007 CP000712.1 Unpublished GB-1 6 078 430 61.9 5515 90.2 74 22 05/02/2008 CP000926.1 Unpublished KT2440 6 181 863 61.5 5481 87.5 74 22 22/01/2003 AE015451.1 Nelson et al. (2002) W619 5 774 330 61.4 5292 89.8 75 22 11/03/2008 CP000949.1 Unpublished P. stutzeri A1501 4 567 418 63.9 4237 90.3 61 13 20/04/2007 CP000304.1 Yan et al. (2008) P. syringae pv. phaseolicola 1448A 5 928 787 57.9 5436‰ 86.8‰ 64 16 01/08/2005 CP000058.1 Joardar et al. (2005a, b) pv. syringae B728a 6 093 698 59.2 5245 88.7 64 16 12/05/2005 CP000075.1 Feil et al. (2005) pv. tomato DC3000 6 397 126 58.3 5721‰ 85.6‰ 64 16 21/08/2003 AE016853.1 Buell et al. (2003)

ÃSize, %G1C from http://www.Pseudomonas.com, apart from PA2192 and C3719 from Mathee et al. (2008). wGenes, % coding, and rRNA and tRNA content from IMG, apart from Pseudomonas fluorescens Pf0-1, SBW-25 and Pf-5 (Silby et al., 2009). zThe genomes of these strains have not been properly deposited in GenBank. ‰Coding sequences from plasmids included in gene count and calculation of % coding. other Pseudomonas spp., P. putida genomes are larger than pathogens. The presence of genes with roles in disease such 6 Mbp. Annotation of the P. putida genome sequences as quorum sensing (QS), alginate biosynthesis and adhesins (Tables 1 and 2) showed a considerable number of CDSs of in KT2440, albeit with some modifications, supports a unknown function, which is typical for Pseudomonas gen- broader role of these genes in the ecological success omes. A comparison of P. putida KT2440 with the P. in addition to their known contributions to the virulence aeruginosa PAO1 genome indicated that 85% of the CDSs of P. aeruginosa. were homologous (Nelson et al., 2002). Interestingly, Pseudomonas putida strains live in diverse soil environ- 508 CDSs (9.4%) were identified as putative duplications, ments. Analysis of the KT2440 genome uncovered features almost double that seen for P. aeruginosa. The largest reflecting the adaptability of P. putida and their applicability family of duplications consists of 41 transposase genes, to bioremediation (Nelson et al., 2002; Weinel et al., 2002). seven of which were novel to P. putida. These data suggest Pseudomonas putida KT2440 encodes 350 outer membrane that gene duplication and transposable elements were of and cytoplasmic transporters (15% more than PAO1) considerable importance in shaping the genome evolution for the uptake/efflux of a wide range of substrates. Also of P. putida. identified were genes predicted to be involved in the By comparison with the genome sequences of P. aerugi- transformation of a range of aromatic compounds not nosa PAO1 and P. syringae, the nonpathogenic lifestyle of previously known to be substrates of KT2440 metabolic P. putida was correlated with the absence of key animal pathways, suggesting that even broader applications of the and plant pathogenicity genes, i.e. KT2440 did not harbour species in bioremediation may be possible. A xenB homo- genes for a type III protein secretion system (T3SS), logue, involved in 2,4,6-trinitrotoluene catabolism, and 12 exotoxin A, alkaline protease, elastase, a rhamnolipid bio- glutathione S-transferases, predicted to be involved in synthesis operon, exolipase or phospholipase C, or genes detoxification, were also identified. KT2440 has 11 LysE specifying plant cell wall-degrading enzymes found in plant family amino acid efflux transporters, compared with one in

Table 2. Genome diversity – predicted genes unique to single strainsÃ but not in the other two strains (Silby et al., 2009). Much of Not present in others the P. fluorescens genomes are asyntenous, indicating a large Pseudomonas isolatew of the speciesz degree of rearrangement. Remarkably, P. fluorescens Pf-5 was P. aeruginosa 2192 187 found to have a very high number (1052) of gap-free 34-bp P. aeruginosa C3719 45 REP sequences, compared with 21 in P. aeruginosa PAO1, P. aeruginosa LESB58 219 365 in P. syringae DC3000 (Tobes & Pareja, 2005) or 804 in P. aeruginosa PA7 660 P. putida KT2440 (Aranda-Olmedo et al., 2002). REP P. aeruginosa PACS2 29 sequences were absent from regions of the Pf-5 genome that P. aeruginosa PAO1 54 encode housekeeping genes, reflecting the strong selection P. aeruginosa UCBPP-PA14 143 pressure for maintenance of function and the dynamically P. fluorescens Pf-5 821

P. fluorescens Pf0-1 657 adaptive variable regions. In KT2440, the distribution of Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 P. fluorescens SBW25 1195 REP sequences was heavily biased toward intergenic spaces, P. putida F1 272 possibly reflecting similar selection pressure (Aranda-Olme- P. putida GB-1 456 do et al., 2002). The genomes of Pf-5, SBW25 and Pf0-1 also P. putida KT2440 422 carry intergenic repeat sequences distinct from the REP P. putida W619 418 sequences, which can be categorized into 12 families ranging P. syringae pv. oryzae str. 1_6 573 in size from 50 to 352 bp (Silby et al., 2009). The distribution P. syringae pv. phaseolicola 1448A 263 P. syringae pv. syringae B728a 216 and density of these repeats vary considerably among the P. syringae pv. tabaci ATCC 11528 353 genomes. The density in SBW25 was sufficiently high to P. syringae pv. tomato DC3000 330 allow the detection of a nonrandom distribution around the P. syringae pv. tomato T1 412 genome, whereby so-called ‘repeat deserts’ lacking repeat sequences were found to cover 40% of the genome. The ÃData retrieved 23 December 2010. wOnly Pseudomonas spp. for which more than one strain has been functional significance of REP and other repeats is not clear, sequenced are shown. but their presence might provide a substrate for recombina- zNumber of genes in a particular isolate not present in other members of tion events that rearrange existing genes and incorporate the same species. newly acquired ones into the genome. In all cases, data were derived from the Joint Genome Institute IMG Like P. putida KT2440, the saprophytic lifestyle of system (http://img.jgi.doe.gov/), using maximum E-value = 1e-5, and P. fluorescens in soil and on plants is reflected in the genome minimum % identity = 30. sequence: the three genomes lack genes for most known virulence factors, although SBW25 carries genes for a T3SS PAO1, indicating that P. putida may need to reduce the levels similar to those found in the pathogenic Pseudomonas of amino acids in the cell to avoid them becoming inhibitory species. Despite the fact that some T3SS genes known to be to growth or that the transporters serve to transport other as important in P. syringae type III secretion are absent, the yet unknown molecules. SBW25 system is expressed in the sugar beet rhizosphere Using the KT2440 genome sequence and a microarray, and is capable of secretion (Rainey, 1999b; Preston et al., the similarity among P. putida strains for which genome 2001; Jackson et al., 2005). No plant disease has been sequences were not available was analysed (Ballerstedt et al., associated with the SBW25 T3SS, consistent with the 2007). Based on the presence/absence of genes, strains were genome-informed assessment that SBW25 harbours a low grouped into two clusters, one of which included a Pseudo- complement of plant-targeting putative type III secreted monas monteilii strain previously thought to be distal to the effectors (T3SE). SBW25 does, however, harbour effector P. putida group. ExoY (Vinatzer et al., 2005) (found in P. aeruginosa), which is thought to target the actin cytoskeleton of eukaryotic cells (Engel & Balachandran, 2009). The T3SS of other Pseudomonas fluorescens -- biocontrol and P. fluorescens strains have been implicated in haemolysis plant growth-promoting bacteria (Sperandio et al., 2010) and for targeting the oomycete Pseudomonas fluorescens are generally regarded as plant pathogen Pythium ultimum (Rezzonico et al., 2005). This commensals and have been studied for their biocontrol and may indicate that SBW25 uses the system against an alter- plant growth promotion properties (Haas & Defago, 2005). native host. It is also possible that SBW25 represents a Pseudomonas fluorescens genomes have a high number of ‘snapshot’ of a strain in the process of losing or acquiring genes with no known function (Tables 1 and 2) (Paulsen the system (Jackson et al., 2005). et al., 2005; Silby et al., 2009). Among the three sequenced P. Genomic analysis has provided an insight into the fluorescens strains, there is a conserved set of 3642 CDSs, and environmental success of P. fluorescens. Each genome en- 1490, 1400 and 1111 CDSs found in Pf-5, SBW25 and Pf0-1, codes a wide array of membrane-based sensors and

c 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 35 (2011) 652–680 Published by Blackwell Publishing Ltd. All rights reserved Genomics of pseudomonads 657 receptors as well as enzymes for diverse catabolic activities, Pseudomonas stutzeri -- a nitrogen-fixing including breakdown of plant-derived substrates such as nonfluorescent pseudomonad lignin (a trait of P. putida). Pf-5 is well known as a producer of secondary metabolites with antimicrobial activity, Pseudomonas stutzeri A1501 survives in soil and colonizes such as the polyketides pyoluteorin (Howell & Stipanovic, plant roots epiphytically and endophytically (Lalucat et al., 1980) and 2,4-diacetylphloroglucinol (Nowak-Thompson 2006). Consistent with its saprophytic lifestyle, pathogeni- et al., 1994), which may be vital for the bacterium to fend city and virulence genes, including those for T3SS and T4SS, off competitors in the soil and plant environment, and are were not found in the genome (Yan et al., 2008). The ability also key targets for exploitation in the biological control of to fix nitrogen means the bacterium has been developed as a plant diseases (Weller, 2007). Examination of the Pf-5 bioinoculant to stimulate plant growth. The P. stutzeri genome is small compared with other Pseudomonas gen- genome sequence suggested sequences that may specify Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 previously undetected toxins (Paulsen et al., 2005). By omes at 4.56 Mbp, encoding just 4146 predicted proteins, focusing on sequences that resemble nonribosomal peptide highlighting the considerable genomic variation that has synthetases or polyketide synthases, a cyclic lipopeptide with been noted in the genus (Tables 1 and 2). The genome lacks activity against oomycete zoospores was discovered (Gross intact prophages, but does contain 42 repeat sequences and et al., 2007). Loper and colleagues used sequence-guided 57 transposases. Four potential GIs were identified based on targeted mutagenesis to functionally analyse a Pf-5 gene an atypical nucleotide composition and association with cluster (rzx) predicted to be responsible for making analo- tRNA genes. Comparison of protein products with the gues of the antifungal/anti-oomycete rhizoxin secondary predicted proteomes of other Pseudomonas species revealed metabolite (Loper et al., 2008). Comparative metabolic the closest relatedness to P. aeruginosa, and analysis of the profiling of wild type and rzx mutant strains confirmed the conserved gene complement among 14 Pseudomonas spp. genome-based prediction of rhizoxin synthesis. Five rhizox- indicates that P. stutzeri is somewhat distantly related to the in analogues were described, one of which was novel, and other sequenced Pseudomonas spp. (Silby et al., 2009), and were shown to have varied toxicity to different plants and shares approximately 56% of protein-coding genes with the nonspecific antitumour activity with human tumour cell nitrogen-fixing bacterium A. vinelandii strain DJ (Setubal lines. The potential use of these newly discovered com- et al., 2009). pounds for biotechnology is obvious, but their contribution Genes most likely to be required for nitrogen fixation and to ecological success is less clear. If they play a role in rhizosphere competence were identified in a 49-kb region competitive fitness, their unique occurrence in Pf-5 among containing 59 genes in P. stutzeri A1501, which, based on the the P. fluorescens isolates suggests a niche-specific activity G1C content, was postulated to be a GI acquired through that may be accomplished by different mechanisms in other horizontal transfer, and inserted between cobS (PST_1301) isolates. and PST_1360 (Yan et al., 2008). The high level of gene A genome-wide functional screen for environmentally synteny in the flanking regions in other Pseudomonas induced loci (EIL) in SBW25 identified 125 sequences that species, coupled with variability between those genes, leads show elevated expression in planta (Silby et al., 2009). to the suggestion that this is a hot spot for insertion or Putative orthologues of 73 of these sequences occur in both variation. Other factors likely to contribute to rhizosphere Pf0-1 and Pf-5, indicating that environmental responses competence include 4 300 transporter genes (87 unique to (and presumably adaptation) are mediated by both con- P. stutzeri), several of which appear to serve for uptake of served and strain-specific factors. The functions of SBW25 dicarboxylic acids, carbohydrates and amino acids that are genes induced in planta and shared among all three strains commonly found in plant root exudates. The genome also include motility, nutrient scavenging, stress response, detox- revealed gene systems likely to be important for success in ification and regulation, each of which would be predicted fluctuating soil environments such as those for the degrada- to be important in natural environments. Genome-wide tion of aromatic compounds, nitrate metabolism, osmoto- screens for regulators of these EIL identified several known lerance, detoxification of reactive oxygen species, and novel regulators, including seven regulators controlling chemotaxis, type IV secretion (for DNA transformation) the expression of a cellulose synthase gene system known to and extracellular polysaccharide (cellulose) production. A be important for plant colonization (Gal et al., 2003; gene for inhibiting ethylene production was also found, Giddens et al., 2007). Intriguingly, several ‘reverse orienta- which could contribute to plant growth promotion by tion’ sequences were also induced in planta in SBW25, reducing ethylene inhibition of root development. Pseudo- suggesting the existence of microRNAs and protein-coding monas stutzeri does not appear to harbour known side- antisense genes similar to cosA and other antisense genes in rophore biosynthesis genes, consistent with its description Pf0-1, which are difficult to predict in genomes (Silby & as nonfluorescent. So how do P. stutzeri survive in iron- Levy, 2004, 2008; Kim et al., 2009). limiting environments? The genome sequence indicates the

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 658 M.W. Silby et al. presence of three putative siderophore receptors (PST_0147, Pseudomonas aeruginosa is a challenging pathogen to 0996 and 4174). Pseudomonas stutzeri may act as a cheat, combat due to the formation of a sessile bioﬁlm during utilizing siderophores produced by other pseudomonads persistent infections (Costerton et al., 1999), which en- (Loper & Henkels, 1999; Harrison & Buckling, 2009). hances resistance to antimicrobial treatments and to host Alternatively, P. stutzeri may have alternative iron acquisi- immune defences (Costerton & Lewandowski, 1995; Dren- tion systems: our analysis of the genome sequence found kard & Ausubel, 2002; Mah et al., 2003). The involvement of that four putative siderophore biosynthesis protein genes three overlapping QS systems (Whiteley et al., 1999; Girard are present in the genome, three of which are clustered & Bloemberg, 2008), a T3SS (Hauser, 2009), sigma factors (PST_2504, 3699, 3701-2). If these were involved in side- (Potvin et al., 2008) and two-component regulatory systems rophore production, they clearly do not confer a ﬂuorescent in controlling virulence and resistance in P. aeruginosa

phenotype. (Gooderham & Hancock, 2009) suggests that numerous Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 virulence factors controlled by these systems may be re- Pathogenicity vealed by functional genomic analysis. PAO1 encodes a large number of regulatory (521), outer Some pseudomonads cause disease in diverse host organ- membrane (150) and cytoplasmic membrane proteins isms. The most studied and economically important species (300), which is consistent with its exposure to diverse are the opportunistic pathogen, P. aeruginosa, and the plant environments. The bacterium encodes a number of trans- pathogen, P. syringae. The economic and health costs porters putatively involved in antibiotic resistance, high- associated with Pseudomonas infections have driven an lighting the significant problems associated with controlling intense interest in their genome sequences. Comparative P. aeruginosa infections. Genes for T1SS, T2SS and T3SS as genomic approaches show the diversity of genes among well as a number of genes for chemotaxis and motility were pathogenic isolates, highlighting the extent of host–patho- also identified. In the original annotation of the PAO1 gen coevolution and the range of mechanisms potentially genome, 2549 (46% of genome) genes were originally used to cause disease in different hosts. annotated to be of unknown function or homologous to genes with unknown function (some of these were reclassi- Pseudomonas aeruginosa -- opportunistic fied; Weinel et al., 2003). This fact almost certainly illustrates human pathogen the diversity of genes that contribute to the complex ecology Pseudomonas aeruginosa is an aquatic and soil bacterium of the bacterium. In November 2010, 2445 genes were still that can infect a range of organisms, including plants classified as having an unknown function, indicating that (Rahme et al., 2000), nematodes (Tan et al., 1999a, b), fruit with the benefit of 10 years of functional research and flies (Apidianakis & Rahme, 2009), waxmoth (Miyata et al., comparative genomics, only around 100 unknown genes 2003), zebrafish (Clatworthy et al., 2009) and various have been functionally characterized. mammals (Snouwaert et al., 1992; Colledge et al., 1995; The size of the PAO1 genome results from genetic Potvin et al., 2003). In humans, P. aeruginosa is an opportu- complexity (more genes of unique function) rather than nistic pathogen capable of infecting a range of tissues and gene duplications. Pseudomonas aeruginosa has a large sites (Lyczak et al., 2000) and is one of the top six infectious number of paralogous groups (distinct gene families), disease threats (Talbot et al., 2006), causing serious infec- indicating that its genome has evolved through genetic tions in patients who are immunocompromised or whose expansion (Stover et al., 2000), which may suggest adapta- natural defences are otherwise breached. For example, it is tion to colonization of and competitive fitness in a diverse associated with infections of burns and wounds patients, range of ecological niches. The function of many genes may and infections of the eye associated with contact lens use become apparent only when analysed within more complex (Robertson & Cavanagh, 2008). Pseudomonas aeruginosa is environments than generally used in laboratories, as has also the major pathogen contributing to the morbidity and been argued in the case of environmentally induced genes in mortality associated with cystic fibrosis (CF), causing P. fluorescens (Rainey, 1999b; Silby & Levy, 2004). chronic lung infections, and makes a considerable contribu- As a means to determine the genomic basis for varied tion to the burden of hospital-acquired infections (Govan & degrees of virulence in different P. aeruginosa isolates, a Deretic, 1996), where it is a frequent cause of respiratory and PAO1 array was constructed and used to evaluate the urinary tract infections, a common cause of hospital- presence and absence of virulence genes in 18 strains of acquired pneumonia, healthcare-associated pneumonia and environmental or clinical origin (Wolfgang et al., 2003). The ventilator-associated pneumonia (American Thoracic So- virulence gene data failed to correlate strongly with an ciety, 2005). Pseudomonas aeruginosa infections are particu- associated phenotypic appraisal of the virulence properties larly challenging because of the organisms’ broad intrinsic of the 18 isolates. Clearly, the combination of variable genes antimicrobial resistance (Lister et al., 2009). in each genome has a considerable impact on pathogenesis.

When used in conjunction with a functional approach (a maintaining core virulence functions. Although there are comprehensive transposon mutant library screen), the gen- 11 named P. aeruginosa GIs (PAGI1-11) (Battle et al., 2009) ome sequence did aid in the identification of common P. in addition to PAI-1 and PAPI-2, it is clear that with each aeruginosa virulence factors as well as PA14-specific viru- new genome sequenced, new GIs can be added to the list. lence genes, but it is striking that Lee et al. (2006) concluded The P. aeruginosa Liverpool Epidemic Strain (LES) is a that it may not be possible to predict the virulence (or particularly successful and aggressive transmissible strain otherwise) of one strain based on the presence (or absence) associated with lung infections in CF patients. The genome of genes known to be important for the pathogenicity of a of the earliest isolated LES genotype, LESB58, was sequenced different strain. with the aim of understanding the genetic basis of its high virulence and its emergence as a significant pathogen

(Winstanley et al., 2009). The LESB58 genome harbours all Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 Comparative analysis of P. aeruginosa genomes except two of the 265 PAO1 virulence genes. However, After PAO1, the genome of P. aeruginosa PA14 was se- significant variations were identified, including duplications quenced. PA14 generally shows greater virulence than of pyoverdine-associated genes and divergence in gene PAO1 and thus provided an opportunity to further explore complement and homology of type IV pili and phenazine the genetic basis for virulence in this species. Ninety-two per biosynthesis genes. Importantly, specific mutations could be cent of the PA14 genome is present in PAO1 and 96% of correlated to phenotypic changes. For example, LESB58 is PAO1 in PA14. The PA14 genome harbours 322 more CDSs nonmotile and analysis of the genome identified a pseudo- than PAO1; strain-specific genes typically cluster together in gene form of gene PA1628, which had previously been found areas of the chromosome exhibiting a low GC content to be important for motility and is thus is likely to be relative to the rest of the chromosome i.e. on probable GIs. responsible for this phenotype. Reduced motility in CF In all, 58 PA14-specific gene clusters (with 478 genes) and 54 isolates is not uncommon (Starkey et al., 2009), although PAO1-specific gene clusters (with 234 genes) were identi- the causes of this adaptation are not always known. A fied. The major virulence-related genomic differences be- mutation in a pseudogene of the GDP-mannose-4,6-hydra- tween PA14 and PAO1 are the presence of two pathogenicity tase gene could be responsible for the rough colony mor- islands in PA14 (He et al., 2004). Remarkably, 63% of the photype of the O-antigen-negative LESB58. It is of PA14-specific CDSs have no known function compared with considerable importance to realize that sufficient genomic 29% for the genome on the whole, suggesting PA14-specific and functional data now exist to allow sequence changes to adaptations to an unknown condition, by uncharacterized be correlated with phenotypic differences. mechanisms. The major difference between the genome of LESB58 and It is clear that GIs and prophages contribute most to other P. aeruginosa genomes was the presence of five GIs and variations among the genomes of P. aeruginosa strains. The six prophages, some of which were novel, while others were genome sequences of three P. aeruginosa strains, PA2192, related to similar elements in other sequenced strains. Signa- PACS2 and C3719, isolated from CF patients have been ture-tagged mutagenesis (STM) demonstrated that genes analysed (Table 1) (Mathee et al., 2008). PA2192 exhibits located in a GI (LES GI-5) and prophages (LES prophages 1, major phenotypic adaptation including conversion to mu- 2, 3, 5) were essential for the competitiveness of this strain in a coidy while C3719, a representative of the Manchester rat model of chronic lung infections (Winstanley et al., 2009), Epidemic Strain (Jones et al., 2002), is known for its showing that the acquisition of unique variable sequences can transmissibility. A comparison of five P. aeruginosa genomes enhance success, and emphasizing the role of bacteriophages (PAO1, PA14, PACS2, PA2192 and C3719) revealed a in the evolution of virulence. relatively large set of 5021 conserved genes, in contrast to The genome sequence of the clinical (nonrespiratory, the modest conserved complement seen in comparative multiresistant) taxonomic outlier P. aeruginosa PA7 revealed analysis of P. fluorescens strains (Silby et al., 2009). Pseudo- considerable divergence from other P. aeruginosa strains monas aeruginosa genomes are organized as mosaics of (Roy et al., 2010). In syntenic regions, the PA7 genome has conserved regions interspersed by insertions containing only 93.5% identity with other sequenced strains (PAO1, blocks of strain-variable genes found in a limited number PA14, LESB58), which generally show 99.5% identity (Spen- of chromosomal locations termed ‘regions of genomic cer et al., 2003). The genome has a similar representation of plasticity’ (RGP) (Mathee et al., 2008). Some of these RGPs functional categories of genes, except for a bias towards are unique to a strain because of the deletion of the gene(s) more DNA replication, repair and recombination genes due in other strains, but many of the strain-specific RGPs have to a high number of transposase and integrase genes in been horizontally acquired. Importantly, the islands inserted GIs. Of the 51 GIs identified, 18 are novel to PA7. Among at RGPs often differ among genomes, indicating the indivi- these islands were genes for heavy metal efflux, ectoine dual evolutionary trajectories of the strains, while also utilization, iron transport, haemagglutinins, fimbrial genes

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 660 M.W. Silby et al. and multidrug efflux genes, representing acquired adaptive infections. Using a rat model for chronic lung infections, traits. The genome sequence was particularly useful for STM was coupled with genome sequence data as a high- unravelling the genetic basis of antibiotic resistance, at throughput screening tool to identify virulence genes (Pot- which PA7 excels: point mutations in gyrA and parC confer vin et al., 2003). This initial study, using PAO1, has since fluoroquinolone resistance and efflux systems with con- been extended to identify in vivo essential genes in the CF served and unique (oprA) components were the primary Liverpool Epidemic Strain (LESB58) (Winstanley et al., modes of resistance. Intriguingly, the PA7 genome lacks the 2009). Of the 47 STM mutants essential for competitiveness genes for the structural T3SS as well as effectors exoS, exoU in a rat model of chronic lung infection, three mapped to and exoY, and several other known virulence genes were different LES prophages and one to a novel GI, demonstrat- absent. Despite the importance of T3SS in some pathogenic ing the importance of the accessory genome in the patho-

P. aeruginosa isolates, it cannot be considered part of the genicity of this strain. A similar approach has been used to Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 conserved core genome of P. aeruginosa. Strains generally identify genes that are important for P. aeruginosa patho- carry the T3SS genes necessary for the secretion of either exoS genicity in Caenorhabditis elegans and mice (Wiehlmann or exoU, and absence of the structural T3SS genes has only et al., 2007a). been reported for PA7. The PA7 genome also highlighted the Numerous studies have exploited the development of limitations of using certain genes in multilocus sequence microarrays, designed based on genome sequence data, in typing (MLST) analyses, with only two of seven commonly order to study gene expression in response to different used genes showing enough conservation to be useful. PA7 is environments, such as during biofilm growth (Whiteley clearly an atypical P. aeruginosa, and some would suggest that et al., 2001) or exposure to oxidative stress (Salunkhe et al., it may represent a different species altogether. 2005). Dotsch et al. (2009b) created a custom-made Affy- The Pseudomonas genome database (http://v2.pseudomo metrix tiling array of 250 000 oligonucleotides of PAO1, nas.com) has played an important role in maintaining covering 85% of the genome to identify single nucleotide updated information via community-based sequence anno- polymorphisms (SNPs) and insertion/deletion events in tation and more recently providing valuable tools for PAO1 and PA14 genomes. It was used to identify mutations comparative genomics (Winsor et al., 2009). in naturally occurring PAO1 mutants that exhibit b-lactam resistance and upregulation of a cepahlosporinase AmpC (Moya et al., 2009). Functional genomics Transposon mutagenesis is a mainstay of bacterial genetic Pseudomonas syringae -- plant pathogen research. In combination with complete genome sequences, additional power is gained by enabling the precise mapping Since P. syringae was first described, similar bacteria have of numerous insertion points, and their relative positions in been isolated from numerous plants showing disease symp- the genome. Both Jacobs et al. (2003) and Lewenza et al. toms. Although once numbering 4 40 species, they are now (2005) created saturated transposon mutant libraries in classified as the single species P. syringae sensu lato (Paller- PAO1. After sequencing and mapping insertion points, oni, 1984): this general classification reflects the lack of Jacobs and colleagues finished with a defined library of adequate taxonomic studies to properly classify the strains. 30 100 unique insertions (five hits per CDS, excluding 678 Indeed, DNA similarity indicates that P. syringae is a very CDSs probably encoding essential functions). Similarly, diverse group and should be considered as a species com- 20 530 unique transposon insertions were mapped within the plex. Depending on their interactions with given plants, PA14 genome (Liberati et al., 2006). Mutants of PAO1 (http:// P. syringae strains are assigned to one of over 50 pathogenic pseudomutant.pseudomonas.com/) and PA14 (http://ausubel variants (pathovars; pvs) (Dye et al., 1980). For example, lab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) are available P. syringae pv. tomato (Pto) infects Solanum lycopersicum L. to the research community. A ‘track’ in GBrowse on the (tomato). Studies of P. syringae have identified several http://v2.pseudomonas.com website facilitates easy identifica- complex networks of interactions between plant defence tion of orthologues of disrupted genes in other sequenced mechanisms and pathogen-associated molecular patterns Pseudomonas spp. Dotsch et al. (2009a) used the PA14 (Jones & Dangl, 2006). The T3SS, common to all pathogenic mutant library to identify genes involved in antibiotic resis- strains, is essential for facilitating the injection of multiple tance, and suggest that data from this functional genomic effector proteins into plant cells to interact with protein approach will allow the rapid prediction of resistance pheno- targets (suppress plant innate immune defences, manipulate types of clinical isolates for which some DNA sequence is hormone signalling and/or elicit cell death) (Cunnac et al., obtained. 2009). The genomes of multiple P. syringae pathovars have STM is a modification of transposon mutagenesis that been, or are being sequenced, with a major goal to use can be used to identify genes that are important during genomic methods to characterize T3SS effector repertoires

c 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 35 (2011) 652–680 Published by Blackwell Publishing Ltd. All rights reserved Genomics of pseudomonads 661 and other features that could contribute to host range and nity to unravel the mechanisms of host range between two specificity (Lindeberg et al., 2008). Pseudomonas syringae also very closely related pathogens. produces phytotoxins during the host–pathogen interaction The P. syringae B728a and 1448A genome sequences have that generally lack host specificity and can directly injure cells provided an insight into two bean pathogens: the brown of both host and nonhost plants (Bender et al., 1999). spot pathogen P. syringae pv. syringae (Psy) B728a and the Pto strain DC3000 is a model P. syringae strain because it halo blight pathogen P. syringae pv. phaseolicola (Pph) also infects Arabidopsis thaliana, a model plant for develop- 1448A (Feil et al., 2005; Joardar et al., 2005a). The Psy ment, physiology and resistance studies. The Pto DC3000 B728a genome is smaller than that of DC3000, and shares genome (Table 1) includes two plasmids of 73 and 67 kb 4273 genes with DC3000 (Feil et al., 2005). At least 375 REP (Buell et al., 2003). The large number of genes of unknown sequences were identified in B728a (cf. 365 in DC3000; 1052

function (Buell et al., 2003) highlights how little is known in P. fluorescens Pf-5; 21 in P. aeruginosa), but only 16 Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 about this species, and the probable complexity of its life insertion sequence (IS) elements were found and unlike under natural environmental conditions. Unlike P. aerugi- DC3000 and 1448A, the strain does not carry a plasmid. nosa and P. putida, Pto DC3000 exhibits a relatively Nine major genomic rearrangements exist between DC3000 high level of gene duplication, with 2735 genes (48%) in and B728a. Many of the novel genes in B728a were on one of 687 paralogous families. 14 GIs, one of which is an integrative and conjugative New genes pertinent to life in plants were discovered element (ICEland) similar to that found in Pph 1302A and in the DC3000 genome. A g-aminobutyric acid (GABA) P. fluorescens Pf-5 (Paulsen et al., 2005; Pitman et al., 2005). permease gene, three GABA transaminase genes and a Although the Pph 1448A genome sequence is smaller than succinate semialdehyde dehydrogenase gene were found B728a (5.92 Mbp), it is more densely coded, with more and predicted to be involved in the utilization of GABA, CDSs spread between one chromosome and two plasmids which is abundant within the apoplast of tomato (Rico & (Joardar et al., 2005a). Like Pto DC3000, the largest class of Preston, 2008). A second important observation was genes in Pph 1448A was categorized as transporters (14%) that Pto DC3000 has a higher number of sugar transporters, and 4 500 (10%) genes were predicted to encode regula- but a lower number of amino acid transporters than tors, emphasizing the ecological and genetic complexity P. aeruginosa PAO1 and P. putida KT2440 (Buell et al., of P. syringae. Between 1448A and DC3000, 77% of CDSs 2003). Several of the sugar transporters are predicted to were syntenic. The identification of 1348 paralogous fa- transfer plant-derived sugars such as xylose, arabinose milies is indicative of a high level of gene duplication in the and ribose. These two observations showed new aspects of genome, similar to DC3000. the P. syringae lifestyle and point to a degree of semi- Psy B728a causes brown spot disease of bean leaves and specialism linked to the pathogen’s ecology. In the DC3000 has been studied extensively in its epiphytic and infectious genome, 298 genes were categorized as virulence factors phases of life (Hirano et al., 1999; Monier & Lindow, 2003, broadly encompassing type III protein secretion, sidero- 2004, 2005). Consistent with its life on the relatively barren phores, phytotoxins, adhesins, extracellular polysaccharides, surface of the leaf, a wide range of genes important for pectic enzymes and detoxification of antimicrobials such as ecological success were predicted and indeed identified, reactive oxygen species and copper, of which PAO1 has 191. including iron-scavenging siderophores, UV resistance, QS, Sixty-five genes were unique to DC3000, and 33 of these copper and antibiotic resistance, osmotolerance and ice were predicted to be type III effectors or helpers. This large nucleation (Marco et al., 2005). Genes likely to be important complement of effectors explains the functional redundancy for virulence after apoplast infection included enzymes to inherent to the T3SS and reflects multiple rounds of patho- target plant tissue (cellulose, pectate lyase, xylanase), auxin gen evolution to overcome host resistance (Stavrinides et al., biosynthesis genes, two toxins (syringomycin and syringo- 2006). The absence of genes for the production of known peptin) and several polyketide/nonribosomal peptide lipodepsinonapeptide phytotoxins such as syringomycin or synthases to make unknown products. The genome se- syringopeptin, which can contribute to virulence (Scholz- quence also revealed 27 putative T3SEs, of which five were Schroeder et al., 2001), hints at mechanistic heterogeneity not found in DC3000 (Feil et al., 2005). Of these, 22 were beyond T3SEs. proven to be secreted by the T3SS (Vinatzer et al., 2006). The genome of a second P. syringae pv. tomato strain, T1, Of the 298 virulence-related CDSs identified in Pto which, unlike DC3000, cannot infect Arabidopsis, aligns with DC3000 (Buell et al., 2003), 81% are shared with 1448A, DC3000 over most of its length. However, 757 CDSs are and 1448A encodes at least 22 experimentally verified T3SEs unique to T1, of which 167 are putative virulence genes (Chang et al., 2005) and potentially up to 34 (Studholme (Almeida et al., 2009). Differences in the T3SE content were et al., 2009); 18 are shared with Pto DC3000. One unusual apparent, with some unique in Pto T1 while some found in feature of the 1448A genome is the presence of two T3SS, the DC3000 were absent. The T1 genome provides an opportu- second of which is probably not involved in plant

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 662 M.W. Silby et al. pathogenicity (Lindgren et al., 1986; Mansﬁeld et al., 1994; zuela et al., 2010). The tumour-inducing olive tree pathogen Joardar et al., 2005a). Because the closest orthologues are in Psv causes olive knot disease. The genome carries 73 genes P. aeruginosa and Photorhabdus, the system may play a role unique to Psv, and has eight major inversions, with two in interactions with different eukaryotic hosts such as postulated to be in Pph after comparison between Pph, Psv insects, nematodes or amoeba. Also of interest in the Pph and Psy B728a (Rodrıguez-Palenzuela´ et al., 2010). The 73 1448A genome was the presence of two T2SS, whereas Pto Psv-speciﬁc genes included those for a range of membrane DC3000 only carries one system: 1448A has more candidate proteins, transporters and regulators. Also discovered were substrates (e.g. cell wall-degrading enzymes) for secretion 12 variable regions, many of which had the features of GIs. than DC3000 (Joardar et al., 2005a). Pph 1448A also encodes One of these contains a region of genes predicted to be three nonribosomal peptide synthase gene clusters including involved in the utilization of anthranilate as the sole carbon

the phaseolotoxin gene cluster, the toxin responsible for and nitrogen source and for the degradation of catechol. Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 causing the chlorotic haloes at sites of infection, but lacks Other genes probably encode proteins for the breakdown of coronatine toxin genes. Altogether, the 1448A genome high- lignin-related aromatic compounds, such as vanillin, to lights the relative similarity between Pto and Pph, but also protocatechuate; genes for protocatechuate metabolism identified novel regions that may be attributable to host were also detected, suggesting that the pathogen has evolved specificity. a system enabling the breakdown of woody tissue of the olive tree. Although 11 genes specifying novel T3SEs were found in Psv, these may not be central to the host specificity. Draft genomes of P. syringae pathovars Rather, the combination of capabilities conferred by the 73 Psv-specific genes may be the critical factors for providing Reinhardt et al. (2009) produced a draft of the rice Psv access to an otherwise impenetrable host. pathogen P. syringae pv. oryzae (Por) genome. Por is The United Kingdom and Western Europe are currently highly diverged from the other P. syringae strains, with only experiencing an epidemic of bleeding canker disease, caused 41 8% identity between the Por and the Pto genomes. by Pae, in European Horse Chestnut trees (Aesculus hippo- The Por genome sequence allowed whittling of the castanum) (Webber et al., 2008; Green et al., 2010). Phylo- conserved set of genes in P. syringae to 3594 CDSs; 97 genes genetic comparison found the Pae strains grouped were found to be unique to Por. The complement of T3SEs with other pathovars of woody hosts, including Psv and (29 effectors were predicted) was found to be unique P. syringae pv. morsprunorum (causes canker in cherry trees). compared with other P. syringae genomes, possibly facilitat- Three genome sequences of European (E-Pae) isolates ing a major goal of P. syringae genomics, determination of and the Indian type strain (I-Pae) that causes leaf spot the extent to which these gene complements define plant symptoms in Aesculus indica were sequenced to study the host range. evolution of the E-Pae strains and to identify the causes for The tobacco pathogen P. syringae pv. tabaci (Pta) genome their sudden emergence in the early 2000s (Green et al., carries 303 CDSs not present in Pph 1448A, Psy B728a or Pto 2010). The E-Pae and I-Pae genomes are 95% similar, with DC3000, and is most closely related to Pph 1448A (97% about 300 kb and 1613 SNPs differing among them. The E- identity) (Studholme et al., 2009). The 24 T3SE genes Pae strains, isolated from different geographic regions of the identified enabled the reappraisal of the core effector British Isles, varied by only three nucleotides and in their complement to just 12 genes common to all P. syringae plasmid content, suggesting that the E-Pae strains share a pathovars. Other differences of possible importance for recent ancestor and were likely derived from a single niche specialization between Pta and Pph were that Pta introduction to Britain. lacked polysaccharide-modifying enzymes and Rhs insecti- The Pae genomes all harbour a T3SS and have an effector cidal toxins, but did carry a tabtoxin cluster that is not content similar to that of Pph 1448A (Green et al., 2010). present in Pph. A total of 102 regions of 4 1 kb were They also carry putative lignin-degradation genes similar to identified as putative Pta GIs. Pta revealed a wide range of those found in Psv, reinforcing the hypothesis that these differences among P. syringae pathovars outside of the systems are important for infection of woody host plants. effector complement that may point the way forward in the Pae isolates carried various haemolysin and filamentous- analysis of adaptations to specific hosts. haemagglutinin genes, as well as xaxAB insect toxin Recent drafts of the genomes of P. syringae pathogens of genes, which could indicate an association with insect tree hosts [P. syringae pv. savastanoi (Psv) NCPPB 3335 (also vectors (Vigneux et al., 2007). Intriguingly, the Pae strains known as P. savastanoi pv. savastanoi; Gardan et al., 1992) harbour a cluster of enterobactin siderophore biosynthesis and P. syringae pv. aesculi e(Pa )] have provided a rapid genes; these are normally found in enteric bacterial patho- insight into the potential mechanisms behind their ability to gens. This is the first discovery of the enterobactin side- infect woody tissues (Green et al., 2010; Rodrıguez-Palen-´ rophore in a pseudomonad, complementing production

c 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 35 (2011) 652–680 Published by Blackwell Publishing Ltd. All rights reserved Genomics of pseudomonads 663 by P. syringae of the more common pyoverdine and 2004). Ferreira et al. (2006) found 119 genes upregulated the less widespread yersiniabactin (Bultreys et al., 2006; (which is in good agreement with Lan et al., 2006) and 76 Jones et al., 2007a; Cornelis, 2010). Because enterobactin is genes downregulated by HrpL. This analysis helped to the most powerful iron-binding siderophore known, it may identify the full regulon controlled by the T3SS regulator as indicate that the host tree, or an alternative host, is well as gain a better understanding of the HrpL cis promoter particularly deﬁcient in available iron. E-Pae strains harbour sequence. In a follow-up experiment, the Lon protease sucrose utilization genes, which are absent from I-Pae, regulon was analysed (Lan et al., 2007) as Lon is known to reﬂecting the pathology of the two pathogens; E-Pae strains lead to the degradation of HrpR and T3SE proteins (Bretz grow in the sucrose-rich vascular systems of tree stems and et al., 2002; Losada & Hutcheson, 2005), and is important branches, while I-Pae is found in leaves, where sucrose is for full virulence of DC3000 in tomato leaves (Lan et al.,

more scarce. 2007). In the DC3000 lon mutant, 155 genes were upregu- Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 The genome of the nonpathogenic P. syringae Psy642 does lated including hrpL and 39 genes in the HrpL regulon, not carry the typical T3SS or the conserved and exchange- while 48 genes were downregulated, including genes for cell able effector loci flanking the hrp/hrc genes (Clarke et al., membrane lipoproteins, extracellular polysaccharides and 2010). However, Psy642 and other phylogenetically similar lipopolysaccharide. The differential levels of expression of nonpathogenic strains can still colonize leaf surfaces and HrpL-regulated genes in the lon mutant indicate that addi- endophytic spaces of plants. Of interest, Psy642 encodes at tional regulatory signals affect their expression. least seven putative effectors including AvrE, ExoYand ExoU homologues, and an atypical T3SS in a different genomic Pseudomonas entomophila -- insect pathogen location. Two genes (hrpK and hrpS) were absent from the cluster. This T3SS is capable of secreting effectors into plant Pseudomonas entomophila was isolated from fruit flies and cells, but the gene cluster was not necessary for growth on was subsequently found to be a pathogen of Drosophila plants. There is some parallel with the T3SS of P. fluorescens (Vodovar et al., 2005). The taxonomic standing of SBW25, i.e. both look like restructured T3SS capable of P. entomophila is unclear, as the species has not been effector secretion, but lack hrpS, have limited effectors described formally (Mulet et al., 2010). Based on the genome including avrE and exoY and are not essential for plant sequence (Tables 1 and 2) (Vodovar et al., 2006), the closest colonization. These secretion systems may serve an alter- relative to P. entomophila is P. putida, with 70% of P. native purpose, perhaps in interactions with nonhost plants entomophila genes present in P. putida: 96% of these are in or insects (Clarke et al., 2010). synteny. The difference in the remaining genes is largely attributed to a high number of paralogous genes in P. putida, rather than a reduction in genome size in P. entomophila Dissection of virulence in P. syringae leading to gene loss. Like several other members of the Bioinformatic analysis of P. syringae genomes has helped in genus, P. entomophila carries numerous REPs of unknown the identification of secretion system genes and their sub- function, distributed widely in the genome (Vodovar et al., strates, which aids in the identification of putative virulence 2006). factors such as the T3SS and T3SEs (Fouts et al., 2002). Two Because of the interest in P. entomophila as a model for transcriptomic studies complemented these analyses using a insect pathogenesis and host–pathogen interactions (Vodo- DC3000 oligo-based array (Lan et al., 2006) or an ORF- var et al., 2005), genome analysis was focused on the based array (Ferreira et al., 2006) to identify genes regulated identification of processes that might contribute to viru- by the T3SS regulators HrpRS and HrpL (the enhancer lence. The gene complement of P. entomophila includes binding proteins HrpRS activate hrpL and the alternate genes that may specify proteins required for survival, transcription factor HrpL binds to hrp box promoters to persistence and immune avoidance in insect hosts and activate T3SS structural genes and effectors). The HrpL systems to produce insecticidal toxins including three regulon generally overlapped the HrpRS regulon, but a TccC-type toxins similar to those of Photorhabdus lumines- number of HrpL-independent genes were identified, indi- cens (ffrench-Constant & Waterfield, 2005) and more dis- cating the more global effect of HrpRS (Lan et al., 2006). As tantly related TccC- and TcdB-type toxins. Also identified well as finding known or predicted T3SS regulated genes, were a putative repeats-in-toxin (RTX) gene and a T1SS other genes not predicted through bioinformatics were unique to P. entomophila, which may be responsible for the identified. Interestingly, housekeeping and flagellum bio- pathogen’s haemolytic activity (Vodovar et al., 2005). Four synthesis genes were downregulated when T3SS genes were lipases, three serine proteases and an alkaline protease upregulated, which is probably an adaptive response similar represent other potentially secreted biologically active pro- to that seen previously (Wolfgang et al., 2004) to prevent teins that could contribute to virulence. Unusually for a triggering of basal immunity in the plant host (Zipfel et al., Gram-negative pathogen, P. entomophila does not encode

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 664 M.W. Silby et al. a T3SS or T4SS; this is particularly interesting, given that the approach, 1300 random clones from a large library derived ‘specialist’ insect pathogen P. luminescens harbours a T3SS from 12 clinical P. aeruginosa strains were examined; 13% important for avoiding phagocytosis (Brugirard-Ricaud contained sequences not found in the PAO1 genome or et al., 2005), as do several strains of the opportunist GenBank (Erdos et al., 2006). There is clearly a greater P. aeruginosa. Although isolation of P. entomophila from a degree of variation among P. aeruginosa isolates than gen- noninsect source has yet to be reported, we suggest that the erally considered, which will become more apparent with abundance of transporter and regulator genes (535 and increased numbers of sequenced genomes. In an interesting 4 300, respectively) reﬂects the probable ecology of the counterpoint to the variability that may exist among pathogen – two lifestyles, one in insects and the other in P. aeruginosa isolates, Dotsch et al. (2010) found that among the environment. This suggestion is in line with the lifestyles 36 clinical isolates, there are 980 genes for which there was

of other Pseudomonas pathogens. absolute conservation at the protein sequence level, and yet Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 only 124 of those could be classified as essential based on Adaptation in environmental and host mutant studies. niches A considerable degree of genetic diversity is apparent in other Pseudomonas species for which more than one isolate Many virulence determinants in P. aeruginosa are generally has been sequenced. For example, P. syringae pv. tabaci viewed as being a part of the conserved gene set (Lee et al., 11528 has 575 CDSs that have no orthologues in P. syringae 2006; Mathee et al., 2008), in contrast to P. syringae, where pv. phaseolicola (most closely related strain) (Studholme effectors are often found linked to mobile DNA such as et al., 2009). Among the three P. fluorescens strains se- transposons, GIs and plasmids (accessory loci). It has been quenced to date, only 3642 genes were conserved (Silby suggested that the content of the accessory genome in et al., 2009), and each isolate carried a large number of CDSs P. aeruginosa determines environmental adaptability such unique to the species (Table 2). The genome-specific genes, as niche expansion (Mathee et al., 2008). However, accessory whether in GIs or not, extend the concept of adaptability genome regions can contribute to virulence (He et al., 2004) and flexibility commonly attributed to pseudomonads. or fitness to establish infections (Winstanley et al., 2009). In addition to the unifying trait of large genomes with a Thus, these differences may reflect two facts: (1) P. aerugi- broad repertoire of functional abilities, different isolates nosa is a more homogeneous pathogen that has emerged have adapted to their particular niches by expanding the relatively recently compared with the P. syringae species pan-genome. complex and (2) P. aeruginosa is a versatile and flexible opportunist capable of causing infections in diverse hosts and at multiple sites within hosts, while P. syringae patho- Inferring lineage-specific adaptation from vars are more specialized. Accessory genes may contribute to genome sequences the increased virulence or competitiveness of particular The ratio of nonsynonymous to synonymous site substitu- strains of P. aeruginosa in specific niches. tions (dN/dS) has been used to evaluate whether positive, relaxed or purifying selection is the predominant selective Diversity in gene content force on genome evolution (Yang & Bielawski, 2000). The While the majority of genes have probable orthologues in more sequences that are available, the more in-depth such other members of the same species, there are a large number analyses. Shapiro & Alm (2009) introduced an additional of unique protein-coding genes in Pseudomonas genomes means by which to measure patterns of selection, based on when compared with closely related isolates (Table 2). the ratio of ‘slow’ vs. ‘fast’ substitutions (S : F); it finds Among P. aeruginosa isolates, Mathee et al. (2008) deter- deviations from a sequence’s ‘normal’ selection regime. mined that there is a conserved set of 5021 genes, but Using these methods, lineage-specific adaptations that between P. aeruginosa isolates, a substantial variation can might have escaped detection because of the absence of be seen. For example, PA7 has 660 CDSs that are absent appropriate experimental data may be revealed, opening from other sequenced P. aeruginosa isolates (maximum new areas of investigation. E-value = 1e-5; minimum % ID = 30; Table 2). Two experi- The S : F ratio is potentially informative when applied to a mental approaches that analysed clone libraries from collec- group of closely related isolates for which whole-genome tions of clinical strains foreshadow what might be expected sequences are available. An elevated S : F at a particular locus when large numbers of P. aeruginosa isolates are sequenced. in one branch of a lineage can point to a specific, possibly Shen et al. (2006) generated a large clone genomic library ecologically relevant, adaptation that the majority of organ- from 12 strains of P. aeruginosa from which 348 (approxi- isms in the lineage has not undergone. S : F is less useful mately 11%) of a set of randomly chosen clones harboured when several members of a lineage are under similar sequences distinct from those found in PAO1. In a similar selective pressures, because it would not be possible to

c 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 35 (2011) 652–680 Published by Blackwell Publishing Ltd. All rights reserved Genomics of pseudomonads 665 classify particular sites as generally ‘slow’ changing if indeed 2009; Jackson et al., 2011). In P. aeruginosa LESB58, 4 75 there is a high degree of variability at those sites. When genes were found on three novel islands. Interestingly, the applied to Pseudomonas genomes, Shapiro & Alm (2009) 110-kb LESGI-3 island showed a bipartite structure, with detected an elevated S : F ratio in some energy production 67 kb identical to PAGI-2, indicating significant island proteins [pyruvate dehydrogenase E1 component (AceE) evolution. One of the novel islands harboured the biosyn- and succinate dehydrogenase complex (SdhCD)], which in thetic genes for the antifungal pyoluteorin, found in some most species tend to have low S : F ratios. Although S : F P. fluorescens strains, while the other two carried a mix of alone cannot discern between positive or relaxed negative regulator, transporter, sensor and restriction–modification selection when a lineage such as Pseudomonas AceE and genes (Winstanley et al., 2009). SdhCD show a high S : F compared with other lineages, it Variants of the plasmid/integrative and conjugative ele-

does potentially indicate ecological adaptation and thus ment (ICE) pKLC102 have been identified in P. syringae Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 allow for the generation of testable hypotheses. For example, B728a and several P. aeruginosa strains. Pseudomonas aeru- Shapiro & Alm (2009) proposed a possible link between the ginosa clone C isolates from the lungs of CF patients and changes in P. fluorescens with growth in oxygen-limiting from aquatic environments (Romling et al., 1994) carry biofilms, which has led to the selection of phenazine variants of pKLC102, often integrated as an island, with compounds as terminal electron acceptors. Moreover, sequence characteristics indicating both plasmid and phage changes in SdhCD may reflect changes in redox balance origins (Klockgether et al., 2004). Genome sequencing has associated with oxidative stress experienced by Pseudomonas revealed related elements in P. aeruginosa strains PA14, PA7, interacting with eukaryotes such as plants. Whether these 2192, C3719, LES and PACS2, but not PAO1. In all data indicate positive selection for an adaptive change or a sequenced pKLC102-like elements, there are homologues of relaxed negative selection remains to be determined. 75–98 of the 103 genes in pKLC102, of which a conserved set of 34 are probably involved in transfer, replication and Genomic Islands, bacteriophage and plasmids -- recombination (Wurdemann¨ & Tummler,¨ 2007). Island- the accessory genome in Pseudomonas spp. specific genes make up the remainder of pKLC102-like GIs. In PA14, the pKLC102-like GI is the pathogenicity island Bacteriophages PAPI-1. The relatedness of strains carrying pKLC102-like elements does not correlate with the relatedness of the Although it seems likely that there is a finite number of elements themselves (Wurdemann¨ & Tummler,¨ 2007), in- phage types in the P. aeruginosa pan-genome, there is often dicating frequent transfer, a possibility that is well supported sequence variation even between related prophages in dif- by experimental evidence of transfer (Qiu et al., 2006; Lovell ferent genomes (Ceyssens & Lavigne, 2010), with prophage et al., 2009) and by the diversity and broad distribution of genomes often exhibiting a mosaic structure. Among the six such elements (Klockgether et al., 2007). phage-related gene clusters in P. aeruginosa strain LES In P. fluorescens strains, genome sequences have revealed (isolate LESB58 with five complete and one defective the diversity of integrated elements. In Pf-5, nine GIs, some phage), only the gene cluster encoding a Pf1-like filamen- of which encode secondary metabolites, and six phage-like tous prophage is shared with PAO1 (Winstanley et al., 2009). regions were identified, some resembling phages found in LES prophages 2 and 3 share a duplicated region with each the enteric Shigella and Yersinia (Mavrodi et al., 2009). The other, share some regions with a previously sequenced phage search for GIs uncovered insertion hotspots, such as be- (F10) and contain novel regions. LES prophage 4 is highly tween the mutS and cinA genes, where P. fluorescens strains related, but not identical to the previously sequenced have prophage-like elements (Mavrodi et al., 2009; Silby phage D3112, while LES prophage 5 contains some regions et al., 2009), and P. putida GB-1, P. syringae DC3000 and similar to another characterized phage, D3, and also shares a P. entomophila have various other genes. A 115-kb ICE duplicated region with LES prophage 3. Hence, there is island (ICEland) PFGI-1 was found inserted into a tRNALYS evidence for duplications, genetic rearrangement and recom- gene on the Pf-5 chromosome similar to an ICEland found bination driving diversity in prophages, and the emergence of in P. syringae (Jackson et al., 2000; Pitman et al., 2005), LES isolates with different combinations of prophages sug- and related elements are present in SBW25 and Pf0-1 gests that prophages contribute to genome instability in LES (Silby et al., 2009), revealing their ubiquity as well as the during infection (Fothergill et al., 2010b). heterogeneity of gene content carried in the cargo regions of the ICElands (Reva et al., 2006; Mavrodi et al., 2009). A GIs P. syringae ICEland (PPHGI-1) carrying a T3SE can excise GIs, encompassing pathogenicity islands and fitness islands, from the P. syringae chromosome during growth within are frequently associated with genes that contribute to resistant plants and transfer to recipient strains by transfor- pathogenicity or fitness (Arnold et al., 2003; Battle et al., mation or conjugation. This can also lead to other genomic

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 666 M.W. Silby et al. changes in the recipient, demonstrating the impact that GIs bination between ISPsy6 insertion sequences. In P. putida, can have on the evolution of new pathogen types (Pitman the surE-mutS region contains an intron-encoding matur- et al., 2005; Lovell et al., 2009, 2010; Godfrey et al., 2010, 2011). ase, another indication that this could be a hotspot for insertion of mobile DNA. Based on their CDS content, the 44 P. syringae DC3000 Plasmids LSRs were grouped into phage, ﬁtness, virulence, extrachro- Autonomously replicating plasmids that do not integrate mosomal and MGEs, and unknown. The virulence LSRs into the host genome have so far been uncommon in the include the hrp T3SS pathogenicity island, the coronatine sequenced Pseudomonas spp. P. syringae isolates DC3000 toxin cluster and islands containing T3SE genes. Several of and 1448A both carry two plasmids, but the plasmids in one these were inserted into tRNA genes, including the 99-kb

strain are quite distinct from those in the other (Joardar LSR10, which is a pKLC102-like element related in part Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 et al., 2005a). Several virulence factor homologues are to the PAPI-1 island of P. aeruginosa and PPHGI-1 of present on the plasmids p1448A-A and p1448A-B, but are P. syringae pv. phaseolicola (He et al., 2004; Pitman et al., found on the DC3000 chromosome. Similarly, most of the 2005). Interestingly, analysis ofT3SEs and hrp box promo- virulence factor genes on DC3000 plasmids are chromoso- ters (part of the type III regulon) found 83% and 85%, mal in 1448A. The virulence factors on p1448A-A include respectively, associated with LSRs. One of the phage LSRs the gene virPphA (hopAB1) essential for virulence in bean also contains T3SEs. The fitness LSRs contained genes for cultivars and an avirulence gene recognized by soybean sucrose utilization, cyanate detoxification, resistance to tell- (Jackson et al., 1999, 2002). However, the PAI lacks avrPphF urite and macrolides, chemotaxis genes and a nitrilase. (hopF1) due to transposase-mediated rearrangement (Tsia- Although nitrilase activity has not been detected in Pto mis et al., 2000; Rivas et al., 2005). The virulence genes on DC3000, a Psy B728a nitrilase enables the utilization of the DC3000 plasmid pDC3000A have chromosomal para- indole-3-acetonitrile and phenylpropionitrile as nitrogen logues, raising the possibility that they are not essential for sources, and also mediates synthesis of the plant hormone the pathogenicity of DC3000 (Buell et al., 2003). The indole acetic acid by the bacterium, which may favour the plasmid pQBR103 (and a variety of other plasmids) was bacterium–plant interaction (Howden et al., 2009). Overall, identified in P. fluorescens SBW25 after the previously this study highlighted the power of comparative genomics to plasmid-free SBW25 was introduced into sugar beet crops identify species-specific genes that may otherwise have been as a seed dressing (Tett et al., 2007). Although the original overlooked through traditional approaches. In strain 1448A, isolate of SBW25 did not carry a plasmid (Silby et al., 2009), almost 5% (256) of CDSs are related to MGEs. Several the acquisition of plasmids such as pQBR103, in which only transposase genes are present, the majority of which differ 20% of CDSs are associated with functional predictions, between the B728a and 1448A genomes, indicating that they demonstrated that there is a considerable pool of elements were acquired after divergence of the two lineages. moving among environmental bacteria in the phytosphere. In their comparison of five P. aeruginosa genomes, Mathee et al. (2008) identified 52 RGPs that can result either from insertions of GIs or phage genomes or from the deletion of Lineage-specific regions (LSR) and Regions of genes from one or more strains. Using subtractive hybridiza- Genomic Plasticity (RGP) tion, Battle et al. (2008, 2009) identified seven putative GIs Joardar et al. (2005b) compared the genomes of DC3000 (termed PAGI-5–11) in the highly virulent P. aeruginosa PSE9 with PAO1 and KT2440 to identify LSRs, many of which that were absent from PAO1. Only three of those GIs were were hypothesized to be involved in fitness or pathogenicity. correlated with RGPs, and the gene content of the islands was This was done by identifying LSRs and features within them different from that found in five sequenced isolates (Mathee associated with horizontal gene transfer such as an altered et al., 2008), indicating that knowledge of P. aeruginosa GIs is G1C content and an atypical nucleotide composition. In not yet complete in terms of both the possible genomic total, 44 LSRs were identified in DC3000, of which 34 are locations and the range of cargo genes carried. larger than 10 kb. Of the 983 CDSs within LSRs, 533 are unique to DC3000. Many CDSs with an atypical trinucleo- Adaptation in the CF lung tide composition or an altered G1C content were found in the LSRs, indicating a correlation between LSRs and lateral Pseudomonas aeruginosa is the most common pathogen gene transfer. Interestingly, parts of the DC3000 chromo- infecting the lungs of CF patients, causing chronic infections some are enriched with LSRs and parts appear to be that contribute to the morbidity and mortality associated preferred sites for genes acquired by horizontal transfer; in with the disease. In contrast to their behaviour in acute particular, the surE-mutS region is associated with a chro- infections, in chronic infections, P. aeruginosa populations mosomal inversion likely catalysed by homologous recom- diversify into multiple coexisting phenotypic variants. These

c 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 35 (2011) 652–680 Published by Blackwell Publishing Ltd. All rights reserved Genomics of pseudomonads 667 manifest themselves in a number of ways, including the 2006). Additional diversification of the PAO1 genome has emergence of colony morphology variants (Govan & Deretic, been detected recently using genome resequencing of sub- 1996; Haussler et al., 2003), hypermutability (Oliver et al., lines derived from the original PAO1 isolate. The changes 2000) and diversity in both antimicrobial susceptibility and include duplication of a prophage region, deletions and virulence traits (von Gotz et al., 2004; Fothergill et al., 2007). SNPs (Klockgether et al., 2010), revealing microevolution in Using genomics to study sequential isolates, it was demon- PAO1 and highlighting the plasticity of the genome. A strated that specific mutations often occur during this process similar inversion phenomenon was seen for the P. syringae of adaptation, and this has led to the suggestion that loss of pv. tomato DC3000 genome, which was attributed to trans- virulence may be advantageous (Smith et al., 2006). However, poson and plasmid variation (Landgraf et al., 2006), and diversity in P. aeruginosa populations is a striking feature of resequencing of the Psy B728a genome identified 11 nucleo-

chronic infections, and some members of the population tide substitutions that may represent errors in the original Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 maintain virulence capabilities even after many years of sequence or mutations that have arisen in the samples infection (Fothergill et al., 2010a). (Farrer et al., 2009). These studies highlight the importance of maintaining isogenic stocks and the speed with which substantial genomic changes can accumulate as bacteria Genome organization adapt to their laboratory environments. When comparing the three completely sequenced Variation in the distribution of ‘conserved’ genes P. fluorescens isolates, extensive reciprocal recombination Not only does gene content vary among isolates, but at about the replication terminus was detected (Silby et al., another level – the genome-wide distribution of ortholo- 2009). In addition, some loci are inverted in one strain gous genes – the diversity and variability of Pseudomonas relative to the others, and numerous other loci have been genomes are apparent. There is considerable flexibility in the translocated, with or without also being inverted (Fig. 3). genomes of pseudomonads, evidenced by the occurrence of The sequences least susceptible to such a rearrangement large inversions and translocations such as those observed are found close to the origin of replication. Similarly, among during long-term infections by P. aeruginosa (Ernst et al., P. aeruginosa isolates, there is a high degree of stability 2003; Kresse et al., 2003). A striking example is the inversion around the replication origin, with more rearrangements of of a 1.7-Mb segment of the sequenced PAO1 genome relative small genomic regions apparent closer to the replication to the PAO1 sibling isolate DSM-1707 (Schmidt et al., 1996, terminus, although it should be noted that the actual 1998), which occurred in a research laboratory sometime sequences into which RGPs are inserted are highly conserved after 1990, and that is also apparent when comparing PAO1 (Mathee et al., 2008). Alignment of complete P. syringae with PA14 (Fig. 2), resulting in the replication terminus genomes indicates translocations and inversions similar to being moved to a position 38.8% of the way around the those seen in P. fluorescens, although there is less recombina- circular chromosome with respect to the origin (Lee et al., tion about the replication terminus. Related groups of Pseudomonas spp. such as P. fluorescens and P. syringae appear to be very tolerant of genetic rearrangement. The underlying processes mediating these rearrangements are not always clear. Numerous repetitive sequences, which could be substrates for homologous recombination, have been identified in P. fluorescens in addition to the rRNA operons (Paulsen et al., 2005; Silby et al., 2009), but the boundaries of many rearrangements do not map to those repeat sequences. In the genomes of P. fluorescens isolates, the synteny and proportion of orthologous genes are high near the origin of replication, but decrease toward the replication terminus. This split is not easily explained, and does not correlate with prophages or ‘atypical’ regions in P. fluorescens genomes (Silby et al., 2009) (Figs 3 and 4). Highly expressed genes may cluster near the replication origin because of increased Fig. 2. Geneplot (http://www.ncbi.nlm.nih.gov/sutils/geneplot.cgi) comparisons showing pairs of orthologous genes for Pseudomonas aeruginosa gene dosage during replication resulting from increased PAO1 and PA14. Each axis shows the number of predicted proteins. Note copy number. Conversely, poorly expressed genes, of which the large inversion, which reflects the reported inversion in the PAO1 horizontally transferred genes are often examples, may genome (Stover et al., 2000). cluster nearer the terminus (Rocha, 2004). The latter point

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 668 M.W. Silby et al. Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021

Fig. 3. Comparison of the complete six-frame translations of the whole-genome sequences of Pseudomonas ﬂuorescens Pf0-1, SBW25 and Pf-5. The analysis was carried out using the ARTEMIS Comparison Tool and computed using TBLASTX. Forward and reverse strands of DNA are shown for each genome (dark grey lines). The red bars between the DNA lines represent individual TBLASTX matches, with inverted matches coloured blue. Note the inversions and translocations, and the reduced number of orthologous pairs near the replication terminus (center of ﬁgure).

Fig. 4. Geneplot comparisons showing pairs of orthologous genes for (a) Pseudomonas fluorescens Pf-5 and Pf0-1 and (b) Pseudomonas syringae B728a and 1448A. The axes show the number of predicted proteins. Close to the origin of replication, there is a high degree of synteny in both species. Note the deterioration of synteny in P. fluorescens genomes near the replication terminus, compared with the P. syringae isolates, which show a high degree of synteny along with a few inversions. would be consistent with the regions of low orthology. Genome-based taxonomy of However, support for bias toward the preferential accumu- Pseudomonas lation of mobile genes at the terminus is weak (Rocha, 2004), and there are few indicators in P. fluorescens that the There are at least two goals for classifying microbial species. terminus region has widespread incorporation of mobile One is to categorize organisms together based on simi- elements (Silby et al., 2009). larity, allowing predictions of behaviour. A second goal is to

c 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 35 (2011) 652–680 Published by Blackwell Publishing Ltd. All rights reserved Genomics of pseudomonads 669 use the relationships among strains and species in an list lacks most of the genes commonly used in phylogenetic evolutionary context, to understand ancestral life and analyses including MLST, highlighting the potential im- the processes that have shaped life as we know it. In provements that can be generated using genome-based short, the questions are where did they come from and analyses (see supporting information in Mathee et al., what do they do now? With knowledge of evolution 2008). The utility of the list for the broader genus remains potential, it may be possible to predict the outcomes of to be determined, and should be tested using the 240 strains projected evolution, for example how a pathogen evolves of diverse origin, which Wiehlmann et al. (2007b) show antibiotic resistance. form two major and multiple minor clonal groups. In the absence of morphological traits and breeding range Bacterial species deﬁnition is a controversial subject barriers that have aided the taxonomic classiﬁcation/species (Staley, 2006). Mutations, inversions and acquisitions all

concept in multicellular sexual creatures, classification sys- act to muddle the various analyses and the borderline Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 tems for bacteria have traditionally depended on phenotypic between species is particularly murky. DNA sequence analy- and biochemical properties, and more recently on small- sis has helped to clarify phylogenetic relationships with scale, but informative molecular information. The power of MLST and multilocus sequence analysis (MLSA) providing the latter is exemplified by the revolutionary reclassification strong discrimination. MLST uses four to 10 gene sequences of life into three kingdoms based on rRNA sequences and alleles are assigned numbers. If two strains have the (Woese & Fox, 1977). The advent of genome-based methods same alleles for the gene set being used, then they are provides an opportunity to examine the relatedness of assigned to one sequence type (ST), whereas differences bacteria at an unprecedented level of molecular detail. result in the assignment of unique STs; analysis of popula- Even at a quick glance, questions regarding the classifica- tions is then based on ST numbers. Conversely, MLSA uses tion of pseudomonads into species groups arise. It is the actual DNA sequences for phylogenetic analysis and it is common for a given isolate of a particular ‘species’ to have considered more powerful for analysis of bacteria with less a gene complement in which 4 8% of the genes have no clearly defined species boundaries (Gevers et al., 2005; orthologues in other members of the ‘species’.While some of Almeida et al., 2010). Guttman et al. (2008) argue that this variation may be accounted for by horizontal gene flow, MLST data based on housekeeping genes provide a strong and thus dismissed as ‘accessory’ information, it is unlikely phylogenetic signal allowing accurate construction of evolu- that all of those genes are the product of recent transfer tionary relationships among pseudomonads. This may be events. In the case of P. fluorescens, there are a large number true, given the relatively low frequency of recombination of unique genes in each strain, but there is little evidence observed for housekeeping genes, but gene selection will that the majority of these were recently acquired, pointing to have a major influence on the outcome because some genes a long-term evolutionary process and highlighting that are in linkage disequilibrium while other core genes appear P. fluorescens is probably a species complex (Silby et al., to be freely recombining (Wiehlmann et al., 2007a). 2009). Genome sequences provide access to all housekeeping genes, Mathee et al. (2008) presented an inferred phylogenetic and with the increased availability of draft genome relationship generated from concatenating 1836 ORFs that sequences, it is probable that robust evolutionary models are common to P. fluorescens and six strains of P. aeruginosa, can be constructed. Constructing MLST-based trees based showing that the multihost pathogen PA14 falls outside the on genome sequence data will provide a key resource for main P. aeruginosa cluster. Within the main cluster, the assessing newly isolated pseudomonads. The growing num- relative distance between branch nodes also suggests sig- ber of genome sequences also makes it possible to sample nificant evolutionary divergence among the remaining more genes for their reliability in phylogenetic profiling, P. aeruginosa strains. It was suggested that this phylogenetic providing guidance to the Pseudomonas community, as was tree for the six P. aeruginosa strains is in agreement with done with Escherichia coli (Konstantinidis et al., 2006). their phenotypes and origins, and that PA14 and PA2192 Average nucleotide identity (ANI) and average amino acid may be phylogenetic outliers due to their larger genome identity (AAI) use pairwise comparisons of isolates based on sizes (Mathee et al., 2008); however, we consider this genes and proteins conserved between particular pairs to unlikely, given that the analysis was restricted to shared determine relationships. Using MLST, ANI and AAI, and a orthologous loci. gene-independent analysis of dinucleotide composition, Mathee et al. (2008) also used a maximum parsimony recent studies have directly examined the relatedness of method to infer phylogenetic relatedness based on concate- different Pseudomonas spp (van Passel et al., 2006; Goris nation of conserved genes. Comparing this tree with in- et al., 2007; Silby et al., 2009; Mulet et al., 2010). Collectively, dividual gene trees led to the selection of a list of 144 these studies indicate that several species of Pseudomonas are phylogenetically useful genes within the species that approx- close to or meet the current expectations of a bacterial imate the whole genome-based phylogeny. Importantly, this species. However, in the case of P. syringae and P. fluorescens,

FEMS Microbiol Rev 35 (2011) 652–680 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 670 M.W. Silby et al. Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021

Fig. 5. AAI for pairs of Pseudomonas spp. While Pseudomonas fluorescens strains do not reach the species cutoff, they are more closely related than SBW25 is to PAO1. Genus and species boundaries are as defined (Konstantinidis & Tiedje, 2007). the situation is less clear. Pseudomonas syringae sensu stricto In terms of overall relatedness, genomic approaches have refers to P. syringae pv. syringae, but the species designation demonstrated the variability among P. fluorescens isolates. has also been applied as a provisional solution to encompass The number of orthologous genes identified as reciprocal all pathovars of the pathogen (Dye et al., 1980). Low DDH best BLAST hits was unexpectedly low, totalling just 59.3% of values between P. syringae strains led to the definition of CDSs in strain Pf-5, and concentrated in 2/3 of the genome nine genomospecies (Gardan et al., 1999). Moreover, it was around the replication origin (Fig. 3). Unique genes com- shown that Pto DC3000 and Psy B728a had low DDH, ANI prised 22–27% of the CDSs in P. fluorescens (Silby et al., and AAI values below the species thresholds and therefore 2009). This is in contrast to P. aeruginosa, which showed that may not be part of the same species (Fig. 5, Goris et al., 2007; depending on the strain, the P. aeruginosa isolates had a Konstantinidis & Tiedje, 2007), suggesting that strains percentage of unique genes ranging from 1.4% to 8.2% classified as P. syringae should be regarded as part of a (Mathee et al., 2008). The P. aeruginosa genome sequences P. syringae species complex. all come from strains isolated from clinical settings, raising the possibility that strain choice may bias our view of the genome. Pseudomonas fluorescens Using dinucleotide frequency as a measure of the genome Pseudomonas fluorescens is a diverse group of pseudomo- signature, van Passel et al. (2006) demonstrated that the nads and has been split into various biovars based on genus Pseudomonas has a slightly above average genomic phenotypes and biochemical activity (Stanier et al., 1966). dissimilarity score, indicating a slightly greater than average Genomic analyses are now confirming these data, and variation within the genus. The dissimilarity score between providing a molecular viewpoint on the basis for this P. fluorescens Pf0-1 and Pf-5 genomes was extremely high, variation. The MLSA analysis by Guttman et al. (2008) suggesting that they might eventually be categorized as using gapA, gltA, gyrB and rpoD demonstrated that the separate species. P. fluorescens clade is very diverse, and actually includes Analysis of three completely sequenced P. fluorescens several other species. There was also lower confidence in the strains using DDH and ANI (Goris et al., 2007) and AAI branching within the P. fluorescens clade than in the other and a phylogenomic approach (Silby et al., 2009) has been clades, as has been seen elsewhere (Yamamoto et al., 2000; particularly revealing. By any of the measures applied, it Silby et al., 2009). Pseudomonas fluorescens trees have similar could be argued that the three P. fluorescens strains should topology whether derived from the shared genome content not be considered as one species. The AAI for P. fluorescens (Silby et al., 2009), eight housekeeping genes (gyrB, rpoB, was approximately 83% (Silby et al., 2009), which is well rpoA, recA, gyrA, rpoD, gltA and gapA) (Rodrıguez-Palen-´ below the species cutoff (Konstantinidis & Tiedje, 2005). zuela et al., 2010), four housekeeping genes (16S rRNA gene, The AAI for other Pseudomonas spp. gets closer to or above gyrB, rpoB and rpoD) (Mulet et al., 2010) or two genes (gyrB the species cutoff, although some P. putida strains fall and rpoD) (Yamamoto et al., 2000). slightly below the species cutoff limit (Fig. 6). However, it

c 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 35 (2011) 652–680 Published by Blackwell Publishing Ltd. All rights reserved Genomics of pseudomonads 671 is not clear whether these data are sufficient to reclassify P. standing the evolutionary history of an organism, and the fluorescens into different species groups. Phylogenomic systematic ordering of those organisms. However, the fact analysis indicates that the P. fluorescens strains are more that these methods necessarily must ignore more variable closely related to each other than they are to other members (or accessory) portions of the genome, where several phe- of the genus, but the different P. fluorescens strains do not notypes of interest are encoded, means that a second goal, reach the species cutoff when compared (Figs 5 and 7). that of functional predictability, cannot always be fully met. It is clear that the evolutionary relationships can be well established using MLST, and ANI/AAI are powerful methods for establishing pairwise relationships. Such techniques Concluding comments allow major goals of taxonomy to be fulfilled – under- There is much that can be learned from the analysis of the linear sequence of nucleotides, when appropriate tools are Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 applied. Carrying out comparative analyses adds further depth to the data, and provides the opportunity to make predictions about the ecological causes for differences in gene complement among related strains and species. The gene predictions from the initial publications are continu- ally being revised, and it should be noted that different informatic approaches yield different conclusions [e.g. P. fluorescens Pf-5 in http://pseudomonas.com (6145 CDSs) and in IMG (6142 CDSs)]. It seems clear that further sequence data will aid in building a better picture of the genus Pseudomonas – in fact, judging by the breadth of mutations and horizontal acquisitions found in many of the described studies, it is evident that the full nature of genomic diversity is far from being realized. With each new completed genome, the estimated conserved gene complement of the genus is revised downward. The conserved complement in five P. aeruginosa isolates was 5021 genes, but when a P. fluorescens strain was included in the analysis, the useable set of genes for phylogenetic analysis declined to Fig. 6. AAI for pairs of Pseudomonas putida isolates. Species boundary is as defined by Konstantinidis & Tiedje (2007). Note that some pairings 1836 (Mathee et al., 2008). In an analysis of 14 Pseudomonas fail to reach the species cutoff. KT2440, W619, GB1 and F1 are P. putida genomes, the common gene set was reduced to 1705 genes strains (see Table 1 for details). (Silby et al., 2009).

Fig. 7. Comparison of conserved (cons) and variable (var) regions in pairs of Pseudomonas fluorescens isolates, as measured by average amino acid. Note that the AAI is considerably lower for conserved sequences in the more variable region than for those sequences in the conserved portion of the P. fluorescens genomes. Genus and species boundaries are as defined (Konstantinidis & Tiedje, 2007).

After 15 years of microbial genome sequencing and A major frontier in the understanding of the biology of analysis, and 10 years of Pseudomonas genome sequencing, Pseudomonas spp. is the exploration of life outside of the we have barely scratched the surface of a complex microbial laboratory, and even in nontraditional niches (Stavrinides world – many of the hypothetical genes of the species are still et al., 2009). A functional genomics approach using emer- a mystery. How do scientists address this issue? We suggest ging sequencing and proteome technology alongside estab- that something as crucial as expression data and phenoarray lished methods, coupled with the ongoing generation of new (metabolomic) analyses (Jones et al., 2007b; Rico & genome sequences, will further our understanding of the Preston, 2008) should be sought as a matter of priority to genus Pseudomonas, and will ensure that we continue to be verify transcriptional activity and identify the signals and surprised by these versatile bacteria. environments that trigger expression. Although the emer-

gence of new and relatively inexpensive sequencing technol- Downloaded from https://academic.oup.com/femsre/article/35/4/652/630861 by guest on 23 September 2021 ogy has already accelerated the rate at which Pseudomonas Acknowledgements genome sequences are being determined, these technologies are not being rapidly used for postgenomic analyses. We thank the three anonymous reviewers for their excellent The use of high-throughput transcriptomic and proteo- suggestions and detailed feedback. M.W.S. and S.B.L. grate- mic technologies should routinely follow genome sequen- fully acknowledge support from the National Research cing to provide a level of experimental verification of gene Initiative Competitive Grant no. 2006-35604-16673 and the predictions. Using microarrays or transcriptome sequen- Agriculture and Food Research Initiative Competitive Grant cing, the presence of transcripts (under specified growth 2010-65110-20392 from the USDA’s National Institute of conditions) can be easily verified and used to support Food and Agriculture, Microbial Functional Genomics computational annotation of the genome (e.g. Yoder-Himes Program. C.W. acknowledges the support of the United et al., 2009; Filiatrault et al., 2010). Transcriptome sequen- Kingdom National Institute for Health Research. cing has the advantage of allowing an experimental analysis of 50 and 30 ends of transcripts, and the identification of transcripts from genes that were not predicted to exist. For References example, analysis of the transcriptome of Helicobacter pylori Almeida NF, Yan S, Lindeberg M et al. (2009) A draft genome using a differential RNA-seq approach revealed considerable sequence of Pseudomonas syringae pv. tomato T1 reveals a type antisense transcription, hundreds of candidate small RNA 0 III effector repertoire significantly divergent from that of genes and several genes for which the 5 end was misanno- Pseudomonas syringae pv. tomato DC3000. Mol Plant-Microbe tated (Sharma et al., 2010). Similarly, proteomic technolo- In 22: 52–62. gies can be used to verify that predicted proteins are actually Almeida NF, Yan S, Cai R et al. (2010) PAMDB, a multilocus made, and identify novel proteins from nonannotated genes sequence typing and analysis database and website for plant- (e.g. Kim et al., 2009). Although these approaches do not associated microbes. Phytopathology 100: 208–215. provide a means by which the existence of a gene can be American Thoracic Society (2005) Guidelines for the disproven, their use in refining genome annotations is likely management of adults with hospital-acquired, ventilator- to increase with the falling costs associated with these associated, and healthcare-associated pneumonia. Am J Resp techniques. Crit Care 171: 388–416. Finally, we strongly suggest that there should be greater Anzai Y, Kim H, Park JY, Wakabayashi H & Oyaizu H (2000) consideration of nonconventional analytical approaches to Phylogenetic affiliation of the pseudomonads based on 16S build our understanding of genomes and to solve many of rRNA sequence. Int J Syst Evol Micr 50: 1563–1589. the unanswered functional genomic questions: the recent Apidianakis Y & Rahme LG (2009) Drosophila melanogaster as a spate of interest in sRNAs (in Pseudomonas, e.g. Kay et al., model host for studying Pseudomonas aeruginosa infection. 2005; Reimmann et al., 2005; Sonnleitner et al., 2009; Nat Protocols 4: 1285–1294. Aranda-Olmedo I, Tobes R, Manzanera M, Ramos JL & Marques Humair et al., 2010) is one marker of evidence that this is S (2002) Species-specific repetitive extragenic palindromic progressing, but other avenues need to be explored further. (REP) sequences in Pseudomonas putida. Nucleic Acids Res 30: While computational annotations are powerful and able to 1826–1833. accurately predict a large number of genes, it is clear that Arnold DL, Pitman A & Jackson RW (2003) Pathogenicity and there are some that escape detection. Using modifications of other genomic islands in plant pathogenic bacteria. Mol Plant the RNA-seq, which generate a strand-specific output, Pathol 4: 407–420. antisense coding regions can be readily detected in bacterial Ballerstedt H, Volkers R, Mars A et al. (2007) Genomotyping of genomes, greatly enhancing the known pool of antisense Pseudomonas putida strains using P. putida KT2440-based genes revealed by genetic means (Silby & Levy, 2004, 2008; high-density DNA microarrays: implications for Silby et al., 2004; Filiatrault et al., 2010; Sharma et al., 2010). transcriptomics studies. Appl Microbiol Biot 75: 1133–1142.

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