The bacterial genus Pseudomonas includes the opportunistic human pathogen P. aeruginosa, plant pathogenic bacteria, plant beneficial bacteria, ubiquitous soil bacteria with bioremediation capabilities and other species that cause spoilage of milk and dairy products. P. aeruginosa can cause chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the ageing population of industrialised societies.
The genome sequences of several pseudomonads have become available in recent years and researchers are beginning to use the data to make new discoveries about this bacterium. This concise volume reviews the most current and topical aspects of Pseudomonas molecular biology and genomics and is aimed at a readership of research scientists, graduate students and other specialists. Renowned international authors have contributed chapters on diverse topics including taxonomy, genome diversity, oligonucleotide usage, polysaccharides, pathogenesis, virulence, biofilms, antibiotic resistance and iron uptake. In addition an entire chapter is devoted to the genetic tools being developed to take full advantage of the wealth of information generated by the genome sequencing efforts. This book is essential reading for anyone involved in Pseudomonas research.
Chapter 1 The Road to the Taxonomy of Pseudomonas
Norberto J. Palleroni
As aptly remarked by one of the anonymous reviewers of this opening chapter, the history of the taxonomy of pseudomonads can be qualified as 'tumultuous'. In fact, the addition of this adjective to the title might make it a bit closer to the point. The studies on the taxonomy of this complicated genus groped their way in the dark while following the classical procedures developed for the description and identification of the organisms involved in sanitary bacteriology during the first decades of the twentieth century. This situation sharply changed with the proposal to introduce as the central criterion the similarities in the composition and sequences of macromolecules components of the ribosomal RNA. The new methodology clearly showed that the genus Pseudomonas, as classical defined, consisted in fact of a conglomerate of genera that could clearly be separated into five so-called rRNA homology groups. Moreover, the taxonomic studies suggested an approach that might proved useful in taxonomic studies of all other prokaryotic groups. A few decades after the proposal of the new genus Pseudomonas by Migula in 1894, the accumulation of species names assigned to the genus reached alarming proportions. At the present moment, the number of species in the current list has contracted more than ten-fold. In fact, this approximated reduction may be even more dramatic if one considers that the present list contains many new names, i.e., relatively few names of the original list survived in the process. The new methodology and the inclusion of approaches based on the studies of conservative macromolecules other than rRNA components, constitutes an effective prescription that helped to reduce Pseudomonas nomenclatural hypertrophy to a manageable size. As indicated in the title, the chapter will emphasize a description of the road ('bumps' included) to an acceptable Pseudomonas taxonomy. As remarked by Roger Stanier in his autobiography, the journey itself, not the arrival, matters. Therefore, as a participant in the journey, I apologize for having included only a limited selection of the contributions taken from the enormous flood of information accumulated on this topic in recent years.
Chapter 2 Genome Diversity of Pseudomonas aeruginosa
Jens Klockgether, Dieco Würdemann, Lutz Wiehlmann, Tim T. Binnewies, David W. Ussery and Burkhard Tümmler
The G+C rich Pseudomonas aeruginosa chromosome consists of a conserved core and a variable accessory part. The core genomes of P. aeruginosa strains are largely collinear, exhibit a low rate of sequence polymorphism and contain few loci of high sequence diversity, notably the pyoverdine locus, the flagellar regulon, pilA and the O-antigen biosynthesis locus. Variable segments are scattered throughout the genome of which about one third are immediately adjacent to tRNA or tmRNA genes. The three known hot spots of genomic diversity are caused by the integration of genomic islands of the pKLC102 / PAGI-2 family into tRNALys or tRNAGly genes. The individual islands differ in their repertoire of metabolic genes, but share a set of syntenic genes that confer their horizontal spread to other clones and species. Colonization of atypical disease habitats predisposes to deletions, genome rearrangements and accumulation of loss-of-function mutations in the P. aeruginosa chromosome. The P. aeruginosa population is characterized by a few dominant clones widespread in disease and environmental habitats. The genome is made up of clone-typical segments in core and accessory genome and of blocks in the core genome with unrestricted gene flow in the population.
Chapter 3 Oligonucleotide Usage Signatures of the Pseudomonas putida KT2440 Genome
Oleg Reva and Burkhard Tümmler
Di- to pentanucleotide usage and the list of the most abundant octa- to tetradecanucleotides are useful measures of the bacterial genomic signature. The Pseudomonas putida KT2440 chromosome is characterized by strand symmetry and intra-strand parity of complementary oligonucleotides. Each tetranucleotide occurs with similar frequency on the two strands. Tetranucleotide usage is biased by G+C content and physicochemical constraints such as base stacking energy, dinucleotide propeller twist angle or trinucleotide bendability. The 105 regions with atypical oligonucleotide composition can be differentiated by their patterns of oligonucleotide usage into categories of horizontally acquired gene islands, multidomain genes or ancient regions such as genes for ribosomal proteins and RNAs. A species-specific extragenic palindromic sequence is the most common repeat in the genome that can be exploited for the typing of P. putida strains. In the coding sequence of P. putida LLL is the most abundant tripeptide.
Chapter 4 Genetic Tools for Pseudomonas
Kyoung-Hee Choi, Lily A. Trunck, Ayush Kumar, Takehiko Mima, RoxAnn R. Karkhoff-Schweizer and Herbert P. Schweizer
Genetic tools are required to take full advantage of the wealth of information generated by genome sequencing efforts, and ensuing global gene and protein expression analyses. Although the development of genetic tools has generally not kept up with the sequencing pace, substantial progress has been made in this arena. PCR- and recombination-based strategies allowed construction of whole genome expression and transposon insertion libraries. Similar strategies combined with improved transformation protocols facilitate high-throughput construction of deletion alleles and development of a broad-host-range mini-Tn7 chromosome integration system. While to date most of these tools and methods have been developed for and applied in P. aeruginosa, they will most likely also be applicable to other Pseudomonas with appropriate modifications.
Chapter 5 Molecular Biology of Cell-Surface Polysaccharides in Pseudomonas aeruginosa: From Gene to Protein Function
Wayne L. Miller and Joseph S. Lam
Cell-surface polysaccharides play diverse roles in the bacterial "lifestyle". They serve as a barrier between the cell wall and the environment, mediate host-pathogen interactions, and form structural components of biofilms. These polysaccharides are synthesized from nucleotide-activated precursors and, in most cases, all the enzymes necessary for biosynthesis, assembly and transport of the completed polymer are encoded by genes organized in dedicated clusters within the genome of the organism. Lipopolysaccharide is one of the most important cell-surface polysaccharides, as it plays a key structural role in outer membrane integrity, as well as being an important mediator of host-pathogen interactions. The genetics for the biosynthesis of the so-called A-band (homopolymeric) and B-band (heteropolymeric) O antigens have been clearly defined, and a lot of progress has been made toward understanding the biochemical pathways of their biosynthesis. The exopolysaccharide alginate is a linear copolymer of ß-1,4-linked D-mannuronic acid and L-guluronic acid residues, and is responsible for the mucoid phenotype of late-stage cystic fibrosis disease. The pel and psl loci are two recently discovered gene clusters that also encode exopolysaccharides found to be important for biofilm formation. Rhamnolipid is a biosurfactant whose production is tightly regulated at the transcriptional level, but the precise role that it plays in disease is not well understood at present. Protein glycosylation, particularly of pilin and flagellin, is a recent focus of research by several groups and it has been shown to be important for adhesion and invasion during bacterial infection.
Chapter 6 Pseudomonas aeruginosa Virulence and Pathogenesis Issues
Victoria E. Wagner, Melanie J. Filiatrault, Kristin F. Picardo and Barbara H. Iglewski
Regulation of gene expression can occur through cell-cell communication or quorum sensing (QS) via the production of small molecules called autoinducers. QS is known to control expression of a number of virulence factors. Another form of gene regulation which allows the bacteria to rapidly adapt to surrounding changes is through environmental signaling. Recent studies have discovered that anaerobiosis can significantly impact the major regulatory circuit of QS. This important link between QS and anaerobiosis has a significant impact on production of virulence factors of this organism. The information provided in this chapter will focus on the more recently identified virulence factors of Pseudomonas, the recent insights into oxygen and its role in pathogenesis, and the issues raised with the molecular biology approaches that have been taken to understand these complex regulatory networks. A comparative overview of available plant and animal models of Pseudomonas which are used to identify virulence factors of this bacterium will also be presented.
Chapter 7 Pseudomonas aeruginosa Biofilms: Impact of Small Colony Variants on Chronic Persistent Infections
Susanne Häußler
The achievements of medical care in industrialised societies are markedly impaired due to chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the ageing population. Chronic infections remain a major challenge for the medical profession and are of great economic relevance because traditional antibiotic therapy is usually not sufficient to eradicate these infections. One major reason for persistence seems to be the capability of the bacteria to grow within biofilms that protects them from adverse environmental factors. Pseudomonas aeruginosa is not only an important opportunistic pathogen and causative agent of emerging nosocomial infections but can also be considered a model organism for the study of diverse bacterial mechanisms that contribute to bacterial persistence. In this context the elucidation of the molecular mechanisms responsible for the switch from planctonic growth to a biofilm phenotype and the role of inter-bacterial communication in persistent disease should provide new insights in P. aeruginosa pathogenicity, contribute to a better clinical management of chronically infected patients and should lead to the identification of new drug targets for the development of alternative anti-infective treatment strategies.
Chapter 8 Antibiotic Resistance in Pseudomonas
Alicia Fajardo and José L. Martínez
Pseudomonas aeruginosa is a highly relevant opportunistic pathogen. One of the most worrisome characteristics of P. aeruginosa consists in its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes and the low permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily develop acquired resistance either by mutation in chromosomally-encoded genes, either by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events that include acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown that phenotypic resistance associated to biofilm formation or to the emergence of small-colony-variants may be important in the response of P. aeruginosa populations to antibiotics treatment. The presence of antibiotic resistance genes in other Pseudomonas species like P. putida, P. fluorescens and P. stutzeri will be discussed as well along this chapter.
Chapter 9 Iron uptake in Pseudomonas
Pierre Cornelis, Christine Baysse and Sandra Matthijs
Like all aerobic bacteria, pseudomonads need to take up iron via the secretion of siderophores which complex iron (III) with high affinity. Much progress has been made in the elucidation of siderophore-mediated high-affinity iron uptake by Pseudomonas, especially in the case of the opportunistic pathogen, P. aeruginosa. Fluorescent pseudomonads produce the high-affinity peptidic siderophore pyoverdine, but also, in many cases, a second siderophore of lesser affinity for iron. Some of the genes for the biosynthesis and uptake of these siderophores have been identified and the functions of the encoded proteins known. Iron uptake via siderophores is regulated at several levels, via the general iron-sensitive repressor Fur (Ferric Uptake Regulator), via extracytoplasmic sigma factors/anti-sigma factors or via other regulators. Since pseudomonads are ubiquitous microorganisms, it is not surprising to find in their genome a large number of genes encoding receptors for the uptake of heterologous ferrisiderophores or heme reflecting their great adaptability to diverse iron sources. Another exciting development is the recent evidence for a cross-talk between the iron regulon and other regulatory networks, including the diffusible signal molecule-mediated quorum sensing in P. aeruginosa.