A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

Aranda, Jesús; Poza, Margarita; Pardo, Belén G.; Rumbo, Soraya; Rumbo, Carlos; Parreira, José Ricardo; Rodríguez-Velo, Patricia; Bou, Germán

doi:10.1186/1471-2180-10-279

Cited by 89 publications

(84 citation statements)

References 26 publications

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“…6A). Accordingly, the basal expression of these genes (A1S_1174-A1S_1173, ddrR, umuDAb, A1S_2008, and A1S_2015) is reduced when the mutant is complemented with the pET-RA plasmid (20) carrying the WT gene (Fig. 6B).…”

Section: Resultsmentioning

confidence: 99%

Identification of a DNA-Damage-Inducible Regulon in Acinetobacter baumannii

et al. 2013

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dThe transcriptional response of Acinetobacter baumannii, a major cause of nosocomial infections, to the DNA-damaging agent mitomycin C (MMC) was studied using DNA microarray technology. Most of the 39 genes induced by MMC were related to either prophages or encoded proteins involved in DNA repair. Electrophoretic mobility shift assays demonstrated that the product of the A. baumannii MMC-inducible umuD gene (umuDAb) specifically binds to the palindromic sequence TTGAAAATGTAAC TTTTTCAA present in its promoter region. Mutations in this palindromic region abolished UmuDAb protein binding. A comparison of the promoter regions of all MMC-induced genes identified four additional transcriptional units with similar palindromic sequences recognized and specifically bound by UmuDAb. Therefore, the UmuDAb regulon consists of at least eight genes encoding seven predicted error-prone DNA polymerase V components and DddR, a protein of unknown function. Expression of these genes was not induced in the MMC-treated recA mutant. Furthermore, inactivation of the umuDAb gene resulted in the deregulation of all DNA-damage-induced genes containing the described palindromic DNA motif. Together, these findings suggest that UmuDAb is a direct regulator of the DNA damage response in A. baumannii. In Escherichia coli and many other bacterial species, extensive DNA damage that cannot be repaired by other cellular mechanisms induces a mutagenic repair pathway known as the SOS response (1). As part of the SOS response, the RecA protein binds to the single-stranded region of the damaged DNA, where it is activated and forms a nucleoprotein filament (2). Activated RecA induces the autoprotease activity of LexA, which in the absence of activated RecA represses the expression of SOS genes by binding to specific sequences in their promoters. As cleaved LexA is unable to bind to these regulator sequences, the SOS genes are derepressed (1). The proteins encoded by SOS genes mediate DNA repair and replication in addition to pausing the cell cycle. The activities in the SOS response include those of highly error-prone polymerases (1). Once the damaged DNA is repaired, RecA no longer induces the autocleavage of LexA, which then accumulates and shuts down the SOS response (3).The SOS genetic network is widely present in Eubacteria (4), but in Acinetobacter spp. the DNA damage response is characterized by several atypical features: (i) there is no damage-induced mutagenesis response to DNA damage, with the remarkable exception of the opportunistic pathogens Acinetobacter baumannii, Acinetobacter ursingii, and Acinetobacter beijerinckii (5-7); (ii) after DNA damage, further induction of recA does not require the RecA protein (8); (iii) none of the promoters of the DNAdamage-inducible genes of Acinetobacter spp. contain a known SOS box (7, 9, 10); (iv) a canonical LexA homologue has not been identified (8, 10, 11); and (v) a UmuD homologue in A. baumannii (UmuDAb) has been proposed as a putative indirect regulator of the DNA damage response (6). Given the...

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Section: Resultsmentioning

confidence: 99%

Identification of a DNA-Damage-Inducible Regulon in Acinetobacter baumannii

et al. 2013

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“…Plasmids were inserted into the target genes as previously described (44). Briefly, the kanamycin and zeocin resistance plasmid pCR-BluntII-TOPO, which is unable to replicate in A. baumannii, served as a suicide vector.…”

Section: Methodsmentioning

confidence: 99%

Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii

et al. 2017

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Acinetobacter baumannii is a major cause of antibiotic-resistant nosocomial infections worldwide. In this study, several rifampin-resistant spontaneous mutants obtained from the A. baumannii ATCC 17978 strain that differed in their point mutations in the rpoB gene, encoding the ␤-subunit of the RNA polymerase, were isolated. All the mutants harboring amino acid substitutions in position 522 or 540 of the RpoB protein were impaired in surface-associated motility and had attenuated virulence in the fertility model of Caenorhabditis elegans. The transcriptional profile of these mutants included six downregulated genes encoding proteins homologous to transporters and metabolic enzymes widespread among A. baumannii clinical isolates. The construction of knockout mutants in each of the six downregulated genes revealed a significant reduction in the surface-associated motility and virulence of four of them in the A. baumannii ATCC 17978 strain, as well as in the virulent clinical isolate MAR002. Taken together, our results provide strong evidence of the connection between motility and virulence in this multiresistant nosocomial pathogen.KEYWORDS Acinetobacter baumannii, surface-associated motility, rpoB, rifampin resistance A cinetobacter baumannii is a Gram-negative bacterium often responsible for nosocomial infections, based on its capacity to acquire and develop antimicrobial resistance (1). It is one of the most frequently encountered pathogens in intensive care, neonatal, and burn units, where it causes pneumonia and infections involving the central nervous system, skin, soft tissues, and bone (1). However, despite the increasing clinical importance of A. baumannii, little is known about the virulence factors that contribute to its pathogenetic properties.In previous studies of DNA damage-mediated mutagenesis in A. baumannii (2-5), we observed an atypical motility pattern in several spontaneous rifampin-resistant (Rif r ) mutants derived from the wild-type (WT) strain ATCC 17978 growing on semisolid medium. Although Acinetobacter spp. lack flagella, they exhibit surface-associated motility (6, 7). In a recent study, hypermotile A. baumannii derivatives with increasing virulence were isolated, but whether a lack of motility negatively affects the virulence and pathogenesis of A. baumannii is unclear (8, 9).The point mutations conferring rifampin resistance are located in the rpoB gene (encoding the ␤-subunit of the RNA polymerase), and they produce substantial changes in the transcriptional profile of bacterial cells by affecting several promoters (3, 10, 11). Nonetheless, not all mutations conferring rifampin resistance give rise to global modifications in the transcriptional profile of the respective bacterium (12, 13). In this RESULTSMotility and virulence of A. baumannii rpoB mutants. By plating saturated cultures of A. baumannii ATCC 17978 in the presence of rifampin (50 mg/liter), we isolated 10 spontaneous mutants (Rif r 1 to Rif r 10) resistant to the antimicrobial. In subsequent motility assays, ...

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“…For fusidic acid, polymyxin B, and rifampin MIC determinations using complemented strains, the medium was supplemented with 10 g/ml of gentamicin (Sigma). (36). For the creation of the lpxC::Km r mutant, a slight modification of this transformation protocol was employed as follows.…”

Section: Methodsmentioning

confidence: 99%

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

et al. 2016

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Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted In most Gram-negative bacteria, many of the proteins responsible for lipopolysaccharide (LPS) synthesis and transport are essential, making them potential targets for antibacterial drug development. In particular, the nine conserved Lpx enzymes are considered promising for the development of novel antibiotics, since (3-deoxy-D-manno-oct-2-ulosonic acid) 2 -lipid A (Kdo 2 -lipid A) is the minimal LPS structure that supports a functional outer membrane (OM) and cell viability for most Gram-negative bacteria (1, 2). Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10-12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).Acinetobacter baumannii is an emerging opportunistic bacterial pathogen of increasing concern due to multidrug resistance (16). A. baumannii is noted for its ability to develop resistance against most conventional antibiotics through mechanisms such as the upregulation of efflux pumps and horizontal transfer of resistance genes (17)(18)(19). Because of this, clinical resistance is becoming a serious issue for this organism (as well as for other Gram-negative pathogens), often necessitating the use of antibiotics of last resort, such as polymyxins (19,20). Understanding resistance to polymyxins, which are cationic and utilize LPS to gain access to cells, has therefore become a focus of renewed in-

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A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

Cited by 89 publications

References 26 publications

Identification of a DNA-Damage-Inducible Regulon in Acinetobacter baumannii

Identification of a DNA-Damage-Inducible Regulon in Acinetobacter baumannii

Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

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