No abstract

Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii, light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptorindependent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle.IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii. In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur. KEYWORDS Acinetobacter baumannii, light, metabolismA cinetobacter baumannii is a threatening human pathogen considered to be the paradigm of multidrug resistance (MDR). A key component in its success as a pathogen is not only its outstanding capability to acquire resistance but also its peculiar

BlsA is a BLUF photoreceptor present in Acinetobacter baumannii, responsible for modulation of motility, biofilm formation and virulence by light. In this work, we have combined physiological and biophysical evidences to begin to understand the basis of the differential photoregulation observed as a function of temperature. Indeed, we show that blsA expression is reduced at 37°C, which correlates with negligible photoreceptor levels in the cells, likely accounting for absence of photoregulation at this temperature. Another point of control occurs on the functionality of the BlsA photocycle itself at different temperatures, which occurs with an average quantum yield of photoactivation of the signaling state of 0.20 ± 0.03 at 15°C < T < 25°C, but is practically inoperative at T > 30°C, as a result of conformational changes produced in the nanocavity of FAD. This effect would be important when the photoreceptor is already present in the cell to avoid almost instantaneously further signaling process when it is no longer necessary, for example under circumstances of temperature changes possibly faced by the bacteria. This complex interplay between light and temperature would provide the bacteria clues of environmental location and dictate/modulate light photosensing in A. baumannii.

Minocycline (MIN) and tigecycline (TIG) are antibiotics currently used for treatment of multidrugresistant nosocomial pathogens. In this work, we show that blue light, as well as white light, modulates susceptibility to these antibiotics in a temperature-dependent manner. The modulation of susceptibility by light depends on the content of iron; an increase in iron results in a reduction in antibiotic susceptibility both under light and in the dark, though the effect is more pronounced in the latter condition. We further provide insights into the mechanism by showing that reduction in susceptibility to MIN and TIG induced by light is likely triggered by the generation of 1 O 2 , which, by a yet unknown mechanism, would ultimately lead to the activation of resistance genes such as those coding for the efflux pump AdeABC. The clinical relevance of these results may lie in surface-exposed wound infections, given the exposure to light in addition to the relatively low temperatures recorded in this type of lesion. We further show that the modulation of antibiotic susceptibility occurs not only in Acinetobacter baumannii but also in other micro-organisms of clinical relevance such as Escherichia coli and Staphylococcus aureus. Overall, our findings allow us to suggest that MIN and TIG antibiotic treatments may be improved by the inclusion of an iron chelator, in addition to keeping the wounds in the dark, a condition that would increase the effectiveness in the control of infections involving these micro-organisms.

NADP-malic enzyme (NADP-ME) catalyzes the oxidative decarboxylation of L-malate, producing pyruvate, CO2 and NADPH. The photosynthetic role of this enzyme in C(4) and Crassulacean acid metabolism (CAM) plants has been well established; however, the biological role of several non-photosynthetic isoforms described in C(3), C(4) and CAM plants is still speculative. In this study, the characterization of the NADP-ME isoforms from Nicotiana tabacum was performed. Three different nadp-me transcripts were identified in this C(3) plant, two of which encode for putative cytosolic isoforms (DQ923118 and EH663836), while the third encodes for a plastidic counterpart (DQ923119). Although the three transcripts are expressed in vegetative as well as in reproductive tissues, they display different levels of expression. With regards to enzyme activity, root is the tissue that displays the highest NADP-ME activity. Recombinant NADP-MEs encoded by DQ923118 and DQ923119 were expressed in Escherichia coli and their kinetic parameters and response to different metabolic effectors were analyzed. Studies carried out with crude extracts and with the recombinant proteins indicate that the cytosolic and plastidic isoforms aggregate as tetramers of subunits of 65 and 63 kDa, respectively. Real-time reverse transcription-PCR studies show that the three nadp-me tobacco transcripts respond differently to several biotic and abiotic stress stimuli. Finally, the physiological role of each isoform is discussed in terms of the occurrence, kinetic properties and response to stress. The structure of the NADP-ME family in tobacco is compared with those of other C(3) species.

Although the physiological and economical relevance of flowers is recognized, their primary metabolism during development has not been characterized, especially combining protein, transcript, and activity levels of the different enzymes involved. In this work, the functional characterization of the photosynthetic apparatus, pigment profiles, and the main primary metabolic pathways were analysed in tobacco sepals and petals at different developmental stages. The results indicate that the corolla photosynthetic apparatus is functional and capable of fixing CO(2); with its photosynthetic activity mainly involved in pigment biosynthesis. The particular pattern of expression, across the tobacco flower lifespan, of several proteins involved in respiration and primary metabolism, indicate that petal carbon metabolism is highest at the anthesis stage; while some enzymes are activated at the later stages, along with senescence. The first signs of corolla senescence in attached flowers are observed after anthesis; however, molecular data suggest that senescence is already onset at this stage. Feeding experiments to detached flowers at anthesis indicate that sugars, but not photosynthetic activity of the corolla, are capable of delaying the senescence process. On the other hand, photosynthetic activity and CO(2) fixation is active in sepals, where high expression levels of particular enzymes were detected. Sepals remained green and did not show signs of senescence in all the flower developmental stages analysed. Overall, the data presented contribute to an understanding of the metabolic processes operating during tobacco flower development, and identify key enzymes involved in the different stages.

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.

Nosocomial infections constitute an important problem in hospitals, intensive care units (ICU) having the highest incidence of this type of infection. Staphylococci, especially Staphylococcus epidermidis and Staphylococcus aureus, are among the most important microorganisms associated with nosocomial infections. S. epidermidis is a common skin resident, and can be introduced into the clinical environment by patients and hospital staff. The situation in hospitals is aggravated by the emergence of multiresistant strains. We evaluated 98 hospital S. epidermidis isolates collected at neonatal, pediatric and adult ICUs and 20 S. epidermidis control skin resident isolates from healthy volunteers, for resistance to ten antibiotics and chemotherapeutic agents, and other pathogenicity factors. A high frequency (76.5%) of multiresistance was detected in clinical isolates, whereas community isolates were resistant to penicillin and ampicillin only. The frequency of multiresistant strains was 67.7% in the neonatal ICU, 66.6% in the pediatric ICU and 60.8% in the adult ICU, the lower frequency of multiresistant isolates in the adult ICU indicates a higher incidence of community strains in this unit. There were significantly higher frequencies of hemolytic, proteolytic and biofilm-forming isolates in the clinical isolates than the community isolates, indicating a higher incidence of strains with pathogenic potential in the hospital environment. Except for slight correlation with hemolytic activity there was no correlation between antibiotic multiresistance and pathogenicity factors.