Acinetobacter baumannii is an opportunistic nosocomial pathogen and one of the six most important multidrug-resistant microorganisms in hospitals worldwide. This human pathogen is responsible for a vast array of infections, of which ventilator-associated pneumonia and bloodstream infections are the most common, and mortality rates can reach 35%. Community-acquired infections have also been reported, but few strains have been recovered from environmental sources and infection reservoirs external to the hospital have not been identified. The majority of A. baumannii infections are caused by two main population clones with worldwide distribution. Infection outbreaks are often associated with multidrug resistance, including the recent emergence of strains resistant to all available antibiotics. Nevertheless, A. baumannii virulence traits and pathogenic potential have mostly remained elusive. The recent expansion of A. baumannii sequenced genomes has permitted the development of large-array phylogenomic and phenotypic analyses, which can offer valuable insights into the evolution and adaptation of A. baumannii as a human pathogen. This review summarises these recent advances, with particular focus on A. baumannii evolutionary and genomic aspects, and proposes new avenues of research.

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A fraction of the nuclear estrogen receptor ␣ (ER␣) is localized to the plasma membrane region of 17␤-estradiol (E2) target cells. We previously reported that ER␣ is a palmitoylated protein. To gain insight into the molecular mechanism of ER␣ residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ER␣ membrane localization. The cancer cell lines expressing transfected or endogenous human ER␣ (HeLa and HepG2, respectively) or the ER␣ nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ER␣ enacts ER␣ association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D 1 promoter activity, DNA synthesis). Moreover, E2 reduces both ER␣ palmitoylation and its interaction with caveolin-1, in a time-and dose-dependent manner. These data point to the physiological role of ER␣ palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation. INTRODUCTIONThe sex steroid 17␤-estradiol (E2) acts by binding to its nuclear receptors (i.e., ER␣ and ER␤) that then transactivate target genes. In addition, E2 induces rapid, nongenomic actions involving plasma membrane-associated signaling that require a membrane ER (Coleman and Smith, 2001;Kelly and Levin, 2001;Jakacka et al., 2002;Marino et al., 2002). Although different structural and functional properties have been reported for the membrane-associated ER by comparison with nuclear ER␣ and ER␤ (Ropero et al., 2002;Toran-Allerand et al., 2002;Deecher et al., 2003), immunocytochemical studies revealed the presence of a significant fraction of nuclear ER also on the plasma membrane (Pappas et al., 1995;Norfleet et al., 1999;Dan et al., 2003;Razandi et al., 2003;Arvanitis et al., 2004;Song et al., 2004). In addition, a single mRNA originates a similarly sized nuclear and membrane ER in ER␣-transfected Chinese hamster ovary and HeLa cells (Razandi et al., 1999;Marino et al., 2002Marino et al., , 2003. Thus, ER␣ localizes to both the nucleus and the plasma membrane. Moreover, the membrane ER␣ is emerging as the primary endogenous mediator of E2 rapid responses important in cell proliferation (Marino et al., 1998(Marino et al., , 2002Castoria et al., 1999Castoria et al., , 2001Razandi et al., 1999Razandi et al., , 2000Lobenhofer et al., 2000;Acconcia et al., 2004a;Fernando and Wimalasena, 2004).Debate is open regarding the structural bases and the mechanisms for ER␣ maintenance at and translocation to the plasma membrane. ER␣ does not display any intrinsic transmembrane domain (Song et al., 2004); thus, ER␣ interaction with specific membrane proteins have been proposed to explain its membrane localization Migliaccio et al., 2002;Razandi et al., 2002Razandi et al., , 2003Toran-Allerand et al., 2002;Arvanitis et al., 2004). In particular, the Ser522 re...

SummaryInducible bacterial amino acid decarboxylases are expressed at the end of active cell division to counteract acidification of the extracellular environment during fermentative growth. It has been proposed that acid resistance in some enteric bacteria strictly relies on a glutamic acid-dependent system. The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB. The gadC gene, located downstream of gadB, has been proposed to encode a putative antiporter implicated in the export of ␥-aminobutyrate, the glutamic acid decarboxylation product. In the present work, we provide in vivo evidence that gadC is co-transcribed with gadB and that the functional glutamic acid-dependent system requires the activities of both GadA/B and GadC. We also found that expression of gad genes is positively regulated by acidic shock, salt stress and stationary growth phase. Mutations in hns, the gene for the histone-like protein H-NS, cause derepressed expression of the gad genes, whereas the rpoS mutation abrogates gad transcription even in the hns background. According to our results, the master regulators H-NS and RpoS are hierarchically involved in the transcriptional control of gad expression: H-NS prevents gad expression during the exponential growth whereas the alternative sigma factor RpoS relieves H-NS repression during the stationary phase, directly or indirectly accounting for transcription of gad genes.

Fig. 3.Organization and evolutionary traits of surface signalling systems of P. aeruginosa and C. crescentus. A. Organisation of fecI (black arrow), fecR (grey arrow) and fecA (white arrow) homologues in the P.aeruginosa (PA) and C. crescentus (CC) genomes. Annotations are according to the P. aeruginosa and C. crescentus genome databases (www.pseudomonas.com and www.tigr.org respectively). Genes (not to scale) are oriented according to the direction of transcription. B. Co-evolution of the iron starvation sigma factors and cognate anti-sigmas. The trees have been adapted from those available at the COG (cluster of orthologous groups of proteins) database (www.ncbi.nlm.nih.gov/cgi-bin/COG). These are based on multiple sequence alignment of all members of COGs 1945 (RpoE orthologues) and 3712 (FecR orthologues), according to the criteria described in www.ncbi.nlm.nih.gov/COG/COGhelp.html. Only protein sequences from P.aeruginosa and C. crescentus were included in the figure. The branch linking the iron starvation subfamily of ECF sigma factors with other members of COG 1945 is indicated (¥). Double-headed arrows connect physically and evolutionarily linked sigma-anti-sigma units.

The whole-genome sequence of an epidemic, multidrug-resistant Acinetobacter baumannii strain (strain ACICU) belonging to the European clone II group and carrying the plasmid-mediated bla OXA-58 carbapenem resistance gene was determined. The A. baumannii ACICU genome was compared with the genomes of A. baumannii ATCC 17978 and Acinetobacter baylyi ADP1, with the aim of identifying novel genes related to virulence and drug resistance. A. baumannii ACICU has a single chromosome of 3,904,116 bp (which is predicted to contain 3,758 genes) and two plasmids, pACICU1 and pACICU2, of 28,279 and 64,366 bp, respectively. Genome comparison showed 86.4% synteny with A. baumannii ATCC 17978 and 14.8% synteny with A. baylyi ADP1. A conspicuous number of transporters belonging to different superfamilies was predicted for A. baumannii ACICU. The relative number of transporters was much higher in ACICU than in ATCC 17978 and ADP1 (76.2, 57.2, and 62.5 transporters per Mb of genome, respectively). An antibiotic resistance island, AbaR2, was identified in ACICU and had plausibly evolved by reductive evolution from the AbaR1 island previously described in multiresistant strain A. baumannii AYE. Moreover, 36 putative alien islands (pAs) were detected in the ACICU genome; 24 of these had previously been described in the ATCC 17978 genome, 4 are proposed here for the first time and are present in both ATCC 17978 and ACICU, and 8 are unique to the ACICU genome. Fifteen of the pAs in the ACICU genome encode genes related to drug resistance, including membrane transporters and ex novo acquired resistance genes. These findings provide novel insight into the genetic basis of A. baumannii resistance.

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl-4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response.

The siderophore pyoverdine (PVD) is a primary virulence factor of the human pathogenic bacterium Pseudomonas aeruginosa, acting as both an iron carrier and a virulence-related signal molecule. By exploring a number of P. aeruginosa candidate systems for PVD secretion, we identified a tripartite ATP-binding cassette efflux transporter, here named PvdRT-OpmQ, which translocates PVD from the periplasmic space to the extracellular milieu. We show this system to be responsible for recycling of PVD upon internalization by the cognate outer-membrane receptor FpvA, thus making PVD virtually available for new cycles of iron uptake. Our data exclude the involvement of PvdRT-OpmQ in secretion of de novo synthesized PVD, indicating alternative pathways for PVD export and recycling. The PvdRT-OpmQ transporter is one of the few secretion systems for which substrate recognition and extrusion occur in the periplasm. Homologs of the PvdRT-OpmQ system are present in genomes of all fluorescent pseudomonads sequenced so far, suggesting that PVD recycling represents a general energysaving strategy adopted by natural Pseudomonas populations.ATP-binding-cassette transporter ͉ periplasm ͉ iron ͉ fluorescent Pseudomonas ͉ secretion