1. The Diversity of the Genus Acinetobacter

Lenie Dijkshoorn and Alexandr Nemec

The genus Acinetobacter comprises 17 validly named and 14 unnamed (genomic) species. Some unrelated (genomic) species have common designations, while some other species seem to be congruent but have different names. The knowledge of the biology or ecology of acinetobacters at species level is limited. This is due to the fact that identification of acinetobacters at species level is difficult. A phenotypic species identification system comprising c. 20 tests has been described (Bouvet and Grimont, 1986) but is not widely used and some closely related species cannot be separated well with this system. A variety of genotypic methods has been explored and applied to investigate the diversity or phylogeny in the genus. These methods include high resolution fingerprinting with AFLP, PCR-RFLP with digestion of PCR amplified sequences, and analysis of various DNA sequences. Of these, AFLP analysis and amplified 16SrDNA ribosomal DNA restriction analysis have been validated with large numbers of strains of all described species. Nucleotide sequence based methods are expected to be the standard for identification in the near future, but a prerequisite for their successful application is the availability of libraries of sequences of strains of all described genomic species. For each species, the sequences should cover the intra-species diversity. Sequence comparisons will also provide a valuable tool to study the phylogenetic relatedness of species.

2. Taxonomy of the Genus Acinetobacter Based on 16S Ribosomal RNA Gene Sequences

Mario Vaneechoutte and Thierry De Baere

The taxonomy of the genus Acinetobacter is complicated and confused. This is not only caused by the numerous and sometimes closely related species which are often impossible to differentiate from each other by phenotypical characteristics (genomic species), but also by microbiologists which have not always been careful in naming new species or in reporting correct data, e.g. when sequencing the 16S rRNA gene. Here we present the current status of the taxonomy of the genus based primarily on the 16S rRNA gene sequences, we discuss briefly the taxonomic position of the 17 nomenspecies, of the 14 genomic species and of several not yet described taxa, we comment on the validity of certain species names, arguing that the recently described A. grimontii is probably synonymous to A. junii, and compare the taxonomic value of sequence determination of the 16S rRNA gene with that of household genes like recA and rpoB for the study of the genus Acinetobacter.

3. Lipopolysaccharides of Acinetobacter

Ralph A. Pantophlet

Lipopolysaccharides (LPS) are the major constituent of the outer membrane of most Gram-negative bacteria. Early structural characterization of Acinetobacter LPS suggested that they may be devoid of an O-polysaccharide, i.e., comprised solely of a core oligosaccharide with a lipid A moiety. In recent years, however, it has become evident that most Acinetobacter LPS do contain an O-polysaccharide chain (O-antigen). Elucidation of the chemical structure of several O-polysaccharides has revealed that they generally are branched and contain amino sugars. Simultaneously, O-polysaccharide-specific monoclonal antibodies have allowed the chemical features of the polymers to be related to serotypes. As such, these antibodies may serve as tools to discriminate Acinetobacter strains based on their O-antigens and to track clinically important clonal groups. Analyses of Acinetobacter LPS core regions have shown that they are structurally different from core regions in most other Gram-negative bacteria, which suggests the possible occurrence of an atypical LPS biosynthetic pathway in Acinetobacter. In addition to enthusiasm for the immunochemical properties and biosynthesis of Acinetobacter LPS, interest in their biological activity has been rekindled recently due to the availability of chemically-defined preparations from clinical strains. Collectively, these studies provide new impulses for investigating the possible roles of LPS in Acinetobacter infection.

4. The Catabolism of Aromatic Compounds by Acinetobacter

Peter A. Williams and Catherine M. Kay

Acinetobacter strains isolated from the environment are capable of the degradation of a wide range of aromatic compounds. However the predominant route for the final stages of assimilation to central metabolites is through catechol or protocatechuate (3,4-dihydroxybenzoate) and the beta-ketoadipate pathway, and the diversity within the genus lies in the channelling of growth substrates, most of which are natural products of plant origin, into this pathway.

5. The Natural Transformation System of Acinetobacter baylyi ADP1: A Unique DNA Transport Machinery

Beate Averhoff and Iris Graf

Natural transformation is a powerful mechanism for generating genetic diversity, evolution of metabolic traits, spreading advantageous alleles and mediating some forms of antigenic variation. Despite the importance of natural transformation the mechanism of uptake of free DNA still remains an obstacle. Natural transformation requires recipient cells in a genetically programmed physiological state known as competence. Acinetobacter sp. strain ADP1, a derivative of the nutritionally versatile soil isolate Acinetobacter sp. BD4, is known for its extraordinary high competence and has become a model strain for investigations of natural transformation systems. Recently, strain ADP1 has been recognized as member of the novel species A. baylyi. Interestingly all members of this species share high competence for natural transformation. The highly efficient natural transformation system of strain ADP1 has forwarded many genetic and physiological studies which gave insights into its nutritional versatility. Moreover, the extraordinary competence and the ease to undergo natural transformation made strain ADP1 a model strain for investigations of natural transformation machineries. Gene transfer studies under environmental conditions and studies on the physiology, bioenergetics, and molecular properties of the ADP1 DNA translocator have forwarded the understanding of the impact of natural transformation on horizontal gene transfer and on structure and function of DNA translocators.

6. Acinetobacter baylyi Genetics

L. Nicholas Ornston, Susan Schlimpert, Alison Buchan and Donna Parke

The ability of some Acinetobacter strains to undergo natural transformation offers extraordinary advantages for genetic analysis. Now recognized as a member of Acinetobacter baylyi, the Acinetobacter strain designated ADP1 exhibits a robust and resilient metabolism that makes it a useful tool for genetic engineering. The sequenced genome of A. baylyi strain ADP1 raises possibilities for analysis of essential genes. Positive selection of loss-of-function, gain-of-function and change-of-function A. baylyi mutants, particularly when coupled to random PCR-mutagenesis and natural transformation, affords a convenient approach for exploration of how structure influences function in a range of important biological molecules.

7. Lysr Homologs in Acinetobacter: Insights into a Diverse and Prevalent Family of Transcriptional Regulators

Sarah H. Craven, Obidimma C. Ezezika, Cory Momany and Ellen L. Neidle

LysR-type transcriptional regulators (LTTRs) comprise the largest family of homologous regulators in prokaryotes. Family members control functions such as biosynthesis and catabolism, host-microbe interactions, virulence, and antibiotic resistance. This chapter focuses on Acinetobacter LTTRs while citing key examples from other bacteria. Although various experimental approaches continue to elucidate features of LysR-type regulation, many questions remain. Structural studies of full-length LTTRs alone and in complex with effectors and target DNA are typically problematic. Recently, the structures of the effector-binding domains of two LTTRs from Acinetobacter baylyi ADP1, BenM and CatM, were characterized. These structures represent the first, and only examples to date, of LTTRs bound to their natural inducers. Comparisons of these structures with and without effectors highlight conformational changes in LTTRs that participate in signal transduction. BenM and CatM control an intricate regulatory circuit for aromatic compound degradation. This regulon illustrates LTTR functions as well as the utility of ADP1 as a model organism. Natural transformation of this strain facilitates powerful genetic experimental approaches. Additionally, this chapter includes an analysis of 44 LTTRs predicted to be encoded by ADP1. Studies of this important regulatory family continue to improve our understanding of microbial metabolism and facilitate the development of biotechnology applications.

8. Spotlight on the Acinetobacter baylyi Beta-Ketoadipate Pathway: Multiple Levels of Regulation

Ulrike Gerischer, Bettina Jerg and Rita Fischer

The beta-ketoadipate pathway is responsible for the degradation of aromatic compounds in Acinetobacter baylyi. To support the catabolism of multiple different substrates, a number of funneling pathways exist. In all cases, the respective genes form clusters and are expressed in operons under the control of specific regulator proteins. These generally recognize the respective aromatic substrate and/or degradation metabolite and bring about an increase in gene expression to provide the necessary set of enzymes. In addition to the specific induction mechanisms exist resulting in a repression of gene expression (despite the continued presence of the aromatic substrate). They respond to the simultaneous presence of other substrates which then are degraded with first priority. These findings indicate that many more genes than known up to now underlie such additional repressing mechanims allowing a more defined gene expression under conditions which are not only defined by one compound but by conditions of multiple carbon sources - a situation probably much closer to the real situation in the environment than a single substrate situation.

9. Potential Application of Acinetobacter in Biotechnology

David L. Gutnick and Horacio Bach

Modern microbiology continues to have a major impact on applied research and development of biotechnology. Many of the characteristics of Acinetobacter ecology, taxonomy, physiology and genetics point to the possibility of exploiting its unique features for future applications. Acinetobacter strains are often ubiquitous, exhibit metabolic versatility, are robust and some provide convenient systems for modern molecular genetic manipulation and subsequent product engineering. In this chapter we explore many of these characteristics and their potential applications including biodegradation and bioremediation, novel lipid and peptide production, enzyme engineering, biosurfactant and biopolymer production and engineering of novel derivatives of these products. It is anticipated that progress in these fields will broaden the range of applications of Acinetobacter for modern biotechnology.

10. Molecular Basis of Acinetobacter Virulence and Pathogenicity

Andrew P. Tomaras, Caleb W. Dorsey, Christin McQueary and Luis A. Actis

Acinetobacter baumannii is the most relevant human pathogen within the Acinetobacter genus. This opportunistic human pathogen causes a wide variety of serious infections in humans, mostly in compromised patients. Recently, A. baumannii has emerged as an important pathogen among wounded soldiers, threatening civilian and military patients. It is apparent that this opportunistic pathogen expresses a myriad of factors that could play a role in the pathogenesis of the infection it causes in humans. Among these factors are the attachment to and persistence on solid surfaces, the acquisition of essential nutrients such as iron, the adhesion to epithelial cells and their subsequent killing by apoptosis, and the production and/or secretion of enzymes and toxic products that damage host tissues. However, very little is known about the molecular nature of most of these processes and factors and almost nothing has been shown with regard to their role in bacterial virulence and the pathogenesis of serious infectious diseases. Fortunately, some of these gaps can now be filled by testing appropriate isogenic derivatives in relevant animal models that mimic the infections in humans, particularly the outcome of deadly pneumonia. Such an approach should provide new and relevant information on the virulence traits of this normally underestimated bacterial human pathogen.

11. Molecular Epidemiology of Acinetobacter species

Harald Seifert and Hilmar Wisplinghoff

Among the three clinically important Acinetobacter species, Acinetobacter baumannii, and unnamed Acinetobacter genomic species 3 and 13TU, A. baumannii is the most significant nosocomial pathogen and predominantly affects patients with impaired host defences in the intensive care unit. It was not until 1986, when major changes in the taxonomy of the genus Acinetobacter and the development of molecular methods to better identify acinetobacters at the species level opened the way for a more detailed study of the epidemiology of these organisms. Some of the methods used for species identification such as ribotyping and AFLP could also be used for strain characterization at the subspecies level. Pulsed-field gel electrophoresis (PFGE) became the gold standard for epidemiological strain typing not only for acinetobacters but for bacteria in general. PCR-based methods giving a lower level of discrimination and reproducibility but easier to perform were devised such as randomly amplified polymorphic DNA-PCR (RAPD-PCR) and repetitive extragenic palindromic (REP) PCR fingerprinting. All these methods are so-called comparative typing methods that require visual or computer-aided side-by-side comparison of molecular fingerprint patterns while multi locus sequence typing (MLST) is a so-called library typing method that was found useful for the study of the population structure of multiple microorganisms including A. baumannii. Using these methods the molecular epidemiology of A. baumannii and of other Acinetobacter species was studied and major insight was gained into the hospital epidemiology of these organisms, their mode of spread, the role of hospital personnel in their transmission and that of environmental surfaces. Spread from one patient to another in the same hospital, spread to another hospital in the same geographical region or spread even to more distantly located regions could be demonstrated. One question that remains to be answered is whether there are a few predominant clonal lineages that are responsible for the epidemic spread of multidrug-resistant A. baumannii within hospitals and across countries.

12. Molecular Basis of Antibiotic Resistance in Acinetobacter spp.

Kevin J. Towner

Members of the genus Acinetobacter have the ability to develop resistance to new antibiotics extremely rapidly. Most multiresistant isolates of Acinetobacter spp. belong to the Acinetobacter baumannii complex, and many clinical isolates of A. baumannii are now resistant to all conventional antimicrobial agents, including carbapenems. Molecular studies have characterised most of the responsible genes and mechanisms of resistance to antibiotics found within the genus. Multidrug resistance typically results from the accumulation of multiple mutations and/or the acquisition of resistance genes from other bacterial genera, with the latter occurring by a variety of mechanisms, including the transfer of plasmids, transposons and integrons carrying clusters of genes encoding resistance to several unrelated families of antibiotics simultaneously. Whole-genome sequence analysis has identified the presence of resistance islands, apparently built through the successive insertion of broad host-range mobile genetic elements into an insertion hotspot on the A. baumannii chromosome. This ability to 'switch' its genetic structure may explain the unmatched speed at which A. baumannii captures resistance markers when under antibacterial selection pressure. Overall, the emergence of resistance among clinical isolates of A. baumannii appears to be a combined effect of gene acquisition, following lateral gene transfer, and clonal spread of multiresistant clones.