Characterization of BLUF-photoreceptors present in Acinetobacter nosocomialis.

Acinetobacter nosocomialis is a Gram-negative opportunistic pathogen, whose ability to cause disease in humans is well recognized. Blue light has been shown to modulate important physiological traits related to persistence and virulence in this microorganism. In this work, we characterized the three Blue Light sensing Using FAD (BLUF) domain-containing proteins encoded in the A. nosocomialis genome, which account for the only canonical light sensors present in this microorganism. By focusing on a light-modulated bacterial process such as motility, the temperature dependence of light regulation was studied, as well as the expression pattern and spectroscopic characteristics of the different A. nosocomialis BLUFs. Our results show that the BLUF-containing proteins AnBLUF65 and AnBLUF46 encode active photoreceptors in the light-regulatory temperature range when expressed recombinantly. In fact, AnBLUF65 is an active photoreceptor in the temperature range from 15°C to 37°C, while AnBLUF46 between 15°C to 32°C, in vitro. In vivo, only the Acinetobacter baumannii BlsA's ortholog AnBLUF65 was expressed in A. nosocomialis cells recovered from motility plates. Moreover, complementation assays showed that AnBLUF65 is able to mediate light regulation of motility in A. baumannii ΔblsA strain at 30°C, confirming its role as photoreceptor and in modulation of motility by light. Intra-protein interactions analyzed using 3D models built based on A. baumannii´s BlsA photoreceptor, show that hydrophobic/aromatic intra-protein interactions may contribute to the stability of dark/light- adapted states of the studied proteins, reinforcing the previous notion on the importance of these interactions in BLUF photoreceptors. Overall, the results presented here reveal the presence of BLUF photoreceptors in A. nosocomialis with idiosyncratic characteristics respect to the previously characterized A. baumannii's BlsA, both regarding the photoactivity temperature-dependency as well as expression patterns, contributing thus to broaden our knowledge on the BLUF family.

Quorum and Light Signals Modulate Acetoin/Butanediol Catabolism in Acinetobacter spp.

Acinetobacter spp. are found in all environments on Earth due to their extraordinary capacity to survive in the presence of physical and chemical stressors. In this study, we analyzed global gene expression in airborne Acinetobacter sp. strain 5-2Ac02 isolated from hospital environment in response to quorum network modulators and found that they induced the expression of genes of the acetoin/butanediol catabolism, volatile compounds shown to mediate interkingdom interactions. Interestingly, the acoN gene, annotated as a putative transcriptional regulator, was truncated in the downstream regulatory region of the induced acetoin/butanediol cluster in Acinetobacter sp. strain 5-2Ac02, and its functioning as a negative regulator of this cluster integrating quorum signals was confirmed in Acinetobacter baumannii ATCC 17978. Moreover, we show that the acetoin catabolism is also induced by light and provide insights into the light transduction mechanism by showing that the photoreceptor BlsA interacts with and antagonizes the functioning of AcoN in A. baumannii, integrating also a temperature signal. The data support a model in which BlsA interacts with and likely sequesters AcoN at this condition, relieving acetoin catabolic genes from repression, and leading to better growth under blue light. This photoregulation depends on temperature, occurring at 23°C but not at 30°C. BlsA is thus a dual regulator, modulating different transcriptional regulators in the dark but also under blue light, representing thus a novel concept. The overall data show that quorum modulators as well as light regulate the acetoin catabolic cluster, providing a better understanding of environmental as well as clinical bacteria.

Through the eyes of a pathogen: light perception and signal transduction in Acinetobacter baumannii.

Sunlight is a ubiquitous environmental stimulus for the great majority of living organisms on Earth; therefore it is logical to expect the development of "seeing mechanisms" which lead them to successfully adapt to particular ecological niches. Although these mechanisms were recognized in photosynthetic organisms, it was not until recent years that the scientific community found out about light perception in chemotrophic ones. In this review we summarize the current knowledge about the mechanism of light sensing through the blue light receptor BlsA in Acinetobacter baumannii. We highlight its function as a global regulator that pleiotropically modulates a large number of physiological processes, many of which are linked to the ability of this opportunist pathogen to persist in adverse intrahospital environments. Moreover, we describe with some specific examples the molecular basis of how this photoregulator senses blue light and translates this physical signal by modulating gene expression of target regulons. Finally, we discuss the possible course of these investigations needed to dissect this complex regulatory network, which ultimately will help us better understand the A. baumannii physiology.

Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens.

Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas.

ISAba825, a functional insertion sequence modulating genomic plasticity and bla(OXA-58) expression in Acinetobacter baumannii.

ISAba825, an insertion sequence found inactivating Acinetobacter baumannii carO, was tagged with a kanamycin (Kn) resistance cassette. ISAba825::Kn effectively transposed in A. baumannii, showing preference for short, AT-enriched target sequences, generating 6- to 9-bp target duplications. Additionally, we detected the presence of ISAba825 upstream of a plasmid-borne bla(OXA-58) gene, generating a hybrid promoter largely enhancing its expression and leading to carbapenem resistance. Overall, a role for ISAba825 in carbapenem resistance modulation in A. baumannii is proposed.

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.

A convenient microbiological assay employing cell-free extracts for the rapid characterization of Gram-negative carbapenemase producers.

OBJECTIVES: The dissemination of metallo and serine carbapenem-hydrolysing beta-lactamases among Gram-negative nosocomial bacteria represents an acute problem worldwide. Here, we present a rapid and sensitive assay for the characterization of carbapenemase producers to aid in infection control and prevention. METHODS: The assay involves a rapid disruption of bacterial isolates with silicon dioxide microbeads, followed by the testing in cell-free extracts of hydrolytic activity towards various beta-lactams including two carbapenems (imipenem and meropenem) and a cephalosporin (ceftazidime). A parallel testing of the effects of selective beta-lactamase inhibitors such as EDTA and clavulanic acid allows differentiation of metallo carbapenemases from serine carbapenemases, and also clavulanic-acid-sensitive from -resistant enzymes among the latter. RESULTS: The efficiency of bacterial disruption using silicon dioxide microbeads was identical to that of ultrasonic treatment. The subsequent microbiological assay aimed to evaluate both substrate specificity and inhibitor profile of carbapenem-hydrolysing enzymes present in the extracts and allowed an accurate differentiation of A, B and D types, as judged by the analysis of 24 well-characterized clinical strains that included metallo-beta-lactamase producers (i.e. VIM-, IMP- and SPM-type Pseudomonas producers; an L1 Stenotrophomonas maltophilia producer; and a GOB-18 Elizabethkingia meningoseptica producer) as well as serine carbapenemase producers (i.e. an SME-type Serratia marcescens producer, a GES-2 Pseudomonas aeruginosa producer, Klebsiella pneumoniae and Citrobacter freundii KPC-2 producers and OXA-type Acinetobacter baumannii producers). CONCLUSIONS: We have developed a convenient microbiological assay aimed to more accurately and in a short time characterize carbapenem-hydrolysing enzymes produced by Gram-negative bacteria. The assay possesses broad applicability in the clinical setting.

Biochemical characterization of metallo-beta-lactamase VIM-11 from a Pseudomonas aeruginosa clinical strain.

A detailed biochemical characterization of the Pseudomonas aeruginosa VIM-11 metallo-beta-lactamase (MbetaL) is reported. The only substitution differentiating VIM-11 from VIM-2 (N165S) promoted a slightly improved catalytic efficiency of the former on 3 out of 12 substrates, notably the bulky cephalosporins. Thus, MbetaL-mediated resistance also may be modulated by remote mutations.

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-[(14)C] ornithine from the medium. L-arginine, and histidine and lysine to a lower extent, could effectively compete for L-[(14)C] 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.

The worldwide spread of metallo-beta-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 beta-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.

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 beta-barrel outer membrane proteins.

The outer membrane proteins responsible for the influx of carbapenem beta-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 beta-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 gamma-Proteobacteria.