Acinetobacter baumannii is a common pathogen whose recent resistance to drugs has emerged as a major health problem. Ethanol has been found to increase the virulence of A. baumannii in Dictyostelium discoideum and Caenorhabditis elegans models of infection. To better understand the causes of this effect, we examined the transcriptional profile of A. baumannii grown in the presence or absence of ethanol using RNA-Seq. Using the Illumina/Solexa platform, a total of 43,453,960 reads (35 nt) were obtained, of which 3,596,474 mapped uniquely to the genome. Our analysis revealed that ethanol induces the expression of 49 genes that belong to different functional categories. A strong induction was observed for genes encoding metabolic enzymes, indicating that ethanol is efficiently assimilated. In addition, we detected the induction of genes encoding stress proteins, including upsA, hsp90, groEL and lon as well as permeases, efflux pumps and a secreted phospholipase C. In stationary phase, ethanol strongly induced several genes involved with iron assimilation and a high-affinity phosphate transport system, indicating that A. baumannii makes a better use of the iron and phosphate resources in the medium when ethanol is used as a carbon source. To evaluate the role of phospholipase C (Plc1) in virulence, we generated and analyzed a deletion mutant for plc1. This strain exhibits a modest, but reproducible, reduction in the cytotoxic effect caused by A. baumannii on epithelial cells, suggesting that phospholipase C is important for virulence. Overall, our results indicate the power of applying RNA-Seq to identify key modulators of bacterial pathogenesis. We suggest that the effect of ethanol on the virulence of A. baumannii is multifactorial and includes a general stress response and other specific components such as phospholipase C.

Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a ␥-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other ␣-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster.The bacterial flagellum is a complex protein structure which consists of a long helical filament that is connected through a flexible linker known as the hook to an H ϩ -or Na ϩ -driven rotary motor (29). Rotation of the filament produces thrust that allows the cell to swim in liquid or semisolid medium (6). Bacteria perform taxis by controlling the frequency of reorientation, which is commonly regulated by a two-component signal transduction system that senses an environmental stimulus (2, 3, 7). Several reorientation mechanisms have been described for different bacteria (35). In Escherichia coli and Salmonella enterica serovar Typhimurium, in which the flagellar system has been studied more extensively, more than 40 proteins are required for flagellar synthesis and functioning (29). Expression of the flagellar genes is regulated in a hierarchical pattern which results from coordination of flagellar gene expression at the transcriptional or posttranscriptional level with one or more structural checkpoints in flagellum biogenesis (31). This tight regulation has probably evolved to avoid unnecessary synthesis of the large number of flagellar protein subunits required for this structure. The high energetic cost required for the synthesis and functioning of the flagellum is compensated for by the selective advantage conferred by motility. Accordingly, motility seems to be important in several processes, such as colonization, pathogenesis, dispersion, and competition for resources (37). When the growth medium is too dense to allow swimming, many bacteria differentiate in...

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