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Many strains of Acinetobacter baumannii, including the highly virulent strain AB5075, undergo a high-frequency switch that results in two cell types that are distinguished by their opaque or translucent colony opacities when viewed by oblique lighting. Opaque (VIR-O) and translucent (AV-T) colonies exhibit multiple phenotypic differences, including virulence. Here we describe how to distinguish between VIR-O and AV-T colony variants and how to generate highly pure stocks of each variant. We also describe methods for measuring opacity switching frequencies of cells grown on agar plates and in liquid cultures.

This chapter outlines a method for making unmarked, in-frame deletion mutations in Proteus mirabilis by allelic replacement. This method utilizes an R6K-based suicide plasmid allowing for integration of the plasmid by homologous recombination via a cloned insert. The plasmid also contains the sacB gene to select for plasmid loss (excision) in the presence of sucrose to create a mutant allele. This method has been applied to the P. mirabilis strains PM7002 and BB2000 and should be generally applicable to other P. mirabilis strains. The same methods described in this chapter can be used to introduce marked or point mutations in a given gene.

Several methods for transposon mutagenesis have been employed for use in P. mirabilis. The first method involves the use of mini-Tn5 derivatives, which are delivered by conjugation of a suicide plasmid containing this transposon, followed by transposition into the chromosome. A second method is the use of preformed transposon/transposase complexes (transposomes), which are introduced into P. mirabilis cells by electroporation. Each of these methods will be discussed along with the advantages and disadvantages of each.

Acinetobacter baumannii cell-cell signaling is mediated by production and release of N-acyl homoserine lactones (AHLs), primarily N-(3-hydroxydodecanoyl)-L-homoserine lactone (3-hydroxy-C 12 -HSL). The secretion of this signal can be readily assessed via simple plate-based or thin-layer chromatography-based assays utilizing an Agrobacterium tumefaciens traG-lacZ containing biosensor. In this chapter, we describe methods to assay for secreted N-acyl homoserine lactone production in Acinetobacter baumannii.

Methods to determine the presence of a bacterial capsule typically involve the use of stains specific for capsule, the exclusion of a dye by the capsule or by electron microscopy. However, these procedures require equipment that may not be readily available to all researchers. Here we describe a method for extraction, visualization, and quantification of extracellular capsular polysaccharide from Acinetobacter baumannii with commonly used reagents and equipment.