The complete sequence of the carbapenem-resistance-conferring conjugative plasmid pLD209 from a Pseudomonas putida clinical strain is presented. pLD209 is formed by 3 well-defined regions: an adaptability module encompassing a Tn402-like class 1 integron of clinical origin containing bla VIM-2 and aacA4 gene cassettes, partitioning and transfer modules, and a replication module derived from plasmids of environmental bacteria. pLD209 is thus a mosaic of modules originating in both the clinical and environmental (nonclinical) microbiota.

The number and type of outer membrane (OM) channels responsible for carbapenem uptake in Acinetobacter are still not well defined. Here, we addressed these questions by using Acinetobacter baylyi as a model species and a combination of methodologies aimed to characterize OM channels in their original membrane environment. Kinetic and competition analyses of imipenem (IPM) uptake by A. baylyi whole cells allowed us to identify different carbapenem-specific OM uptake sites. Comparative analyses of IPM uptake by A. baylyi wild-type (WT) cells and ΔcarO mutants lacking CarO indicated that this OM protein provided a carbapenem uptake site displaying saturable kinetics and common binding sites for basic amino acids compatible with a specific channel. The kinetic analysis uncovered another carbapenem-specific channel displaying a somewhat lower affinity for IPM than that of CarO and, in addition, common binding sites for basic amino acids as determined by competition studies. The use of A. baylyi gene deletion mutants lacking OM proteins proposed to function in carbapenem uptake in Acinetobacter baumannii indicated that CarO and OprD/OccAB1 mutants displayed low but consistent reductions in susceptibility to different carbapenems, including IPM, meropenem, and ertapenem. These two mutants also showed impaired growth on L-Arg but not on other carbon sources, further supporting a role of CarO and OprD/OccAB1 in basic amino acid and carbapenem uptake. A multiple-carbapenem-channel scenario may provide clues to our understanding of the contribution of OM channel loss or mutation to the carbapenem-resistant phenotype evolved by pathogenic members of the Acinetobacter genus.KEYWORDS Acinetobacter, antibiotic resistance, basic amino acid channels, carbapenem outer membrane channels, carbapenem resistance, Gram-negative bacteria, outer membrane proteins T he genus Acinetobacter (family Moraxellaceae, order Pseudomonadales, class Gammaproteobacteria) is composed of Gram-negative aerobic bacteria ubiquitously found in the environment and endowed with a large spectrum of metabolic capabilities (1-5). Some Acinetobacter members, such as those composing the A. calcoaceticus/A. baumannii (Acb) complex, are frequently associated with opportunistic nosocomial infections, with the responsible lineages generally displaying multidrug-resistant (MDR) phenotypes (3, 4). Infectious Acinetobacter lineages have shown an outstanding ability to rapidly evolve resistance when subjected to new antimicrobial challenges, and a

The OmpW family consists of a ubiquitous group of small outer membrane (OM) β-barrel proteins of Gram-negative bacteria with proposed roles in environmental adaptation but poorly understood mechanisms of expression. We report here that Escherichia coli K-12 OmpW contents are drastically modified by temperature changes compatible with the leap from the environment to warm-blooded hosts and/or vice versa. Thus, while OmpW is present in the OM of bacteria grown at 37 °C, it sharply disappears at 23 °C with the concomitant acquisition of colicin S4 resistance by the cells. ompW::lacZY fusions indicated that temperature regulation operates at the level of transcription, being ompW expression almost abolished at 23 °C as compared to 37 °C. Moreover, E. coli Δhns mutants lacking H-NS showed reductions in ompW transcription and OmpW contents at 37 °C, indicating positive modulatory roles for this nucleoid-structuring protein in ompW expression. Also, ΔhnsΔstpA double mutants simultaneously lacking H-NS and its paralog StpA showed more severe reductions in ompW expression at 37 °C, resulting in the complete loss of OmpW. The overall results indicate that OmpW contents in E. coli are regulated by both temperature and H-NS and reinforce OmpW functions in bacterial adaptation to warm-blooded hosts.

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Metallo-␤-lactamases (MBLs) are Zn 2؉-containing secretory enzymes of clinical relevance, whose final folding and metal ion assembly steps in Gram-negative bacteria occur after secretion of the apo form to the periplasmic space. In the search of periplasmic factors assisting MBL biogenesis, we found that dacD null (⌬dacD) mutants of Salmonella enterica and Escherichia coli expressing the pre-GOB-18 MBL gene from plasmids showed significantly reduced resistance to cefotaxime and concomitant lower accumulation of GOB-18 in the periplasm. This reduced accumulation of GOB-18 resulted from increased accessibility to proteolytic attack in the periplasm, suggesting that the lack of DacD negatively affects the stability of secreted apo MBL forms. Moreover, ⌬dacD mutants of S. enterica and E. coli showed an altered ability to develop biofilm growth. DacD is a widely distributed low-molecular-mass (LMM) penicillin binding protein (PBP6b) endowed with low DD-carboxypeptidase activity whose functions are still obscure. Our results indicate roles for DacD in assisting biogenesis of particular secretory macromolecules in Gram-negative bacteria and represent to our knowledge the first reported phenotypes for bacterial mutants lacking this LMM PBP.␤-Lactamase production represents a common mechanism of bacterial resistance and cause of failure in the treatment of infections (1). Among the different groups of ␤-lactamases defined on the basis of structural similarity and catalytic mechanisms, the metallo-␤-lactamases (MBLs) are especially worrisome (2, 3). This group is constituted entirely of metalloenzymes employing Zn 2ϩ for catalysis; members of this group can hydrolyze a broad spectrum of substrates, including the latest generations of clinically relevant ␤-lactams, and can be rapidly spread by horizontal gene transfer (1-3). Among the different strategies for controlling MBL dissemination, efforts have so far been focused on the design of a general MBL inhibitor, a goal so far hindered by the diversity of active-site structures among these metalloenzymes (4).A less known aspect of MBL research, but one that may provide potential targets for antimicrobial drug design, is the MBL biogenesis pathway (5). We previously reported that the productive biogenesis of the GOB-18 MBL in Escherichia coli requires an "expanded" DnaK chaperone system to assist the cytoplasmic transit of the preapoprotein to the secretion system (5). Moreover, we also found that secretion of the apo GOB-18 form is driven by the Sec (SecA-SecYEG) machinery, implying that final folding and Zn 2ϩ ion assembly to the native MBL conformation occur in the periplasm (5). Unfolded proteins emerging from the Sec channel are greeted by a highly complex macromolecule-crowded environment containing an array of modifying enzymes, some of them endowed with the ability to act as folding assistants (6-8). This complex scenario can certainly affect individual folding landscapes (6), but whether the final steps of MBL folding and Zn 2ϩ assembly are assisted by periplasmic ...

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We report here the draft genome sequence of an NDM-1-producing Acinetobacter bereziniae clinical strain, HPC229. This strain harbors both plasmid and chromosomal resistance determinants toward different β-lactams and aminoglycosides as well as several types of multidrug efflux pumps, most likely representing an adaptation strategy for survival under different environments.

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