Alejandro M. Viale

Acquisition of Resistance to Carbapenems in Multidrug-Resistant Clinical Strains of Acinetobacter baumannii: Natural Insertional Inactivation of a Gene Encoding a Member of a Novel Family of β-Barrel Outer Membrane Proteins

ABSTRACT The outer membrane proteins responsible for the influx of carbapenem β-lactam antibiotics in the nonfermentative gram-negative pathogen Acinetobacter baumannii are still poorly characterized. Resistance to both imipenem and meropenem in multidrug-resistant clinical strains of A. baumannii is associated with the loss of a heat-modifiable 29-kDa outer membrane protein, designated CarO. The chromosomal locus containing the carO gene was cloned and characterized from different clinical isolates. Only one carO copy, present in a single transcriptional unit, was found in the A. baumannii genome. The carO gene encodes a polypeptide of 247 amino acid residues with a typical N-terminal signal sequence and a predicted transmembrane β-barrel topology. Its absence from different carbapenem-resistant clinical isolates of A. baumannii resulted from the disruption of carO by distinct insertion elements. The overall data thus support the notion that CarO participates in the influx of carbapenem antibiotics in A. baumannii. Moreover, database searches identified the presence of carO homologs only in species of the genera Acinetobacter, Moraxella, and Psychrobacter, disclosing the existence of a novel family of outer membrane proteins restricted to the family Moraxellaceae of the class γ-Proteobacteria.

Regulation of glycogen metabolism in yeast and bacteria

Microorganisms have the capacity to utilize a variety of nutrients and adapt to continuously changing environmental conditions. Many microorganisms, including yeast and bacteria, accumulate carbon and energy reserves to cope with the starvation conditions temporarily present in the environment. Glycogen biosynthesis is a main strategy for such metabolic storage, and a variety of sensing and signaling mechanisms have evolved in evolutionarily distant species to ensure the production of this homopolysaccharide. At the most fundamental level, the processes of glycogen synthesis and degradation in yeast and bacteria share certain broad similarities. However, the regulation of these processes is sometimes quite distinct, indicating that they have evolved separately to respond optimally to the habitat conditions of each species. This review aims to highlight the mechanisms, both at the transcriptional and at the post-transcriptional level, that regulate glycogen metabolism in yeast and bacteria, focusing on selected areas where the greatest increase in knowledge has occurred during the last few years. In the yeast system, we focus particularly on the various signaling pathways that control the activity of the enzymes of glycogen storage. We also discuss our recent understanding of the important role played by the vacuole in glycogen metabolism. In the case of bacterial glycogen, special emphasis is placed on aspects related to the genetic regulation of glycogen metabolism and its connection with other biological processes.

Evolutionary Relationships among Eubacterial Groups as Inferred from GroEL (Chaperonin) Sequence Comparisons

The essential GroEL proteins represent a subset of molecular chaperones ubiquitously distributed among species of the eubacterial lineage, as well as in eukaryote organelles. We employed these highly conserved proteins to infer eubacterial phylogenies. GroEL from the species analyzed clustered in distinct groups in evolutionary trees drawn by either the distance or the parsimony method, which were in general agreement with those found by 16S rRNA comparisons (i.e., proteobacteria, chlamydiae, bacteroids, spirochetes, firmicutes [gram-positive bacteria], and cyanobacteria-chloroplasts). Moreover, the analysis indicated specific relationships between some of the aforementioned groups which appeared not to be clearly defined or controversial in rRNA-based phylogenetic studies. For instance, a monophyletic origin for the low-G+C and high-G+C subgroups among the firmicutes, as well as their specific relationship to the cyanobacteria-chloroplasts, was inferred. The general observations suggest that GroEL proteins provide valuable evolutionary tools for defining evolutionary relationships among the eubacterial lineage of life.

The Opportunistic Human Pathogen Acinetobacter baumannii Senses and Responds to Light

Light is a ubiquitous environmental signal that many organisms sense and respond to by modulating their physiological responses accordingly. While this is an expected response among phototrophic microorganisms, the ability of chemotrophic prokaryotes to sense and react to light has become a puzzling and novel issue in bacterial physiology, particularly among bacterial pathogens. In this work, we show that the opportunistic pathogen Acinetobacter baumannii senses and responds to blue light. Motility and formation of biofilms and pellicles were observed only when bacterial cells were incubated in darkness. In contrast, the killing of Candida albicans filaments was enhanced when they were cocultured with bacteria under light. These bacterial responses depend on the expression of the A. baumannii ATCC 17978 A1S_2225 gene, which codes for an 18.6-kDa protein that contains an N-terminal blue-light-sensing-using flavin (BLUF) domain and lacks a detectable output domain(s). Spectral analyses of the purified recombinant protein showed its ability to sense light by a red shift upon illumination. Therefore, the A1S_2225 gene, which is present in several members of the Acinetobacter genus, was named blue-light-sensing A (blsA). Interestingly, temperature plays a role in the ability of A. baumannii to sense and respond to light via the BlsA photoreceptor protein.

The chaperone connection to the origins of the eukaryotic organelles

The heat‐shock 60 proteins (Hsp60) constitute a subset of molecular chaperones essential for the survival of the cell, present in eubacteria as well as in eukaryotic organelles. Here, we have employed these highly conserved proteins for the inferences of the origins of the organelles. Hsp60s present in mitochondria from different eukaryotic lineages formed a clade, which showed the closest relationship to that of the Ehrlichia/Rickettsia cluster among the α‐Proteobacteria. This, in addition to phenotypic characteristics, suggests that these obligate intracellular parasites and the lineage that generated the mitochondrion shared last common ancestry. In turn, Hsp60s present in chloroplasts from plants and a red alga, respectively, clustered specifically with those of the cyanobacteria, suggesting that all plastids derive exclusively from this eubacterial lineage.

Loss of a 29-Kilodalton Outer Membrane Protein in Acinetobacter baumannii Is Associated with Imipenem Resistance

ABSTRACT We analyzed the possible causes of imipenem (IPM) resistance in multidrug-resistant isolates of Acinetobacter baumannii. Comparison of the outer membrane protein (OMP) profiles of two genomically related strains (Ab288 [IPM sensitive] and Ab242 [IPM resistant]) indicated the conspicuous loss of a 29-kDa polypeptide in the Ab242 strain. No carbapenemase activity was detected in any of these strains. The treatment of Ab288 with sodium salicylate resulted in IPM resistance and the loss of the 29-kDa OMP. In addition, IPM-resistant clones of Ab288 which were selected by repetitive culturing in increasing concentrations of this antibiotic also showed the absence of this 29-kDa OMP.

Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli

To understand the biological function of bacterial glycogen phosphorylase (GlgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression. glgP deletion mutants (DeltaglgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in DeltaglgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by DeltaglgP cells. The overall results thus show that GlgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.

CarO, an Acinetobacter baumannii outer membrane protein involved in carbapenem resistance, is essential for l-ornithine uptake

We previously associated the emergence of carbapenem resistance in Acinetobacter baumannii with the loss of an outer membrane (OM) protein designated CarO. CarO was found essential for l‐ornithine uptake: CarO‐deficient strains were specifically impaired to grow only on l‐ornithine, and failed to incorporate l‐[14C] ornithine from the medium. l‐arginine, and histidine and lysine to a lower extent, could effectively compete for l‐[14C] ornithine uptake. l‐ornithine also reduced A. baumannii sensitivity to imipenem, suggesting that both compounds compete for uptake. The overall results indicate that CarO participates in the selective uptake of l‐ornithine, carbapenems, and other basic amino acids in A. baumannii.

Sensitive EDTA-based microbiological assays for detection of metallo-{beta}-lactamases in nonfermentative gram-negative bacteria.

ABSTRACT The worldwide spread of metallo-β-lactamase (MBL)-producing gram-negative bacilli represents a great concern nowadays. Sensitive assays for their specific detection are increasingly demanded to aid infection control and to prevent their dissemination. We have developed a novel microbiological assay employing crude bacterial extracts, designated EDTA-imipenem microbiological assay (EIM), to identify MBLs in nonfermentative gram-negative clinical strains. We also evaluated the ability of EIM to detect MBLs in comparison to those of other currently employed screening methods, such as the EDTA disk synergy test (EDS) with imipenem as a substrate and the Etest method. The sensitivities of EIM and Etest were similar (1 versus 0.92, respectively) and much higher than that of EDS (0.67). Moreover, both EIM and Etest displayed the maximum specificity. Modifications were introduced to EDS, including the simultaneous testing of three different β-lactams (imipenem, meropenem, and ceftazidime) and two different EDTA concentrations. This resulted in a sensitivity improvement (0.92), albeit at a cost to its specificity. A simple strategy to accurately detect MBL producers is proposed; this strategy combines (i) an initial screening of the isolates by the extended EDS assay to select the potential candidates and (ii) confirmation of the true presence of MBL activity by EIM.

The Metallo-β-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs.