BlsA Is a Low to Moderate Temperature Blue Light Photoreceptor in the Human Pathogen Acinetobacter baumannii.

Light is an environmental signal that produces extensive effects on the physiology of the human pathogen Acinetobacter baumannii. Many of the bacterial responses to light depend on BlsA, a bluelight using FAD (BLUF)-type photoreceptor, which also integrates temperature signals. In this work, we disclose novel mechanistic aspects of the function of BlsA. First, we show that light modulation of motility occurs only at temperatures lower than 24°C, a phenotype depending on BlsA. Second, blsA transcript levels were significantly reduced at temperatures higher than 25°C, in agreement with BlsA protein levels in the cell which were undetectable at 26°C and higher temperatures. Also, quantum yield of photo-activation of BlsA (lBlsA) between 14 and 37°C, showed that BlsA photoactivity is greatly compromised at 25°C and absent above 28°C. Fluorescence emission and anisotropy of the cofactor together with the intrinsic protein fluorescence studies suggest that the FAD binding site is more susceptible to structural changes caused by increments in temperature than other regions of the protein. Moreover, BlsA itself gains structural instability and strongly aggregates at temperatures above 30°C. Overall, BlsA is a low to moderate temperature photoreceptor, whose functioning is highly regulated in the cell, with control points at expression of the cognate gene as well as photoactivity.

Genetic and Phenotypic Features of a Novel Acinetobacter Species, Strain A47, Isolated From the Clinical Setting.

In 2014, a novel species of Acinetobacter, strain A47, determined to be hospital-acquired was recovered from a single patient soft tissue sample following a traumatic accident. The complexity of the Acinetobacter genus has been established, and every year novel species are identified. However, specific features and virulence factors that allow members of this genus to be successful pathogens are not well understood. Utilizing both genomic and phenotypic approaches, we identified distinct features and potential virulence factors of the A47 strain to understand its pathobiology. In silico analyses confirmed the uniqueness of this strain and other comparative and sequence analyses were used to study the evolution of relevant features identified in this isolate. The A47 genome was further analyzed for genes associated with virulence and genes involved in type IV pili (T4P) biogenesis, hemolysis, type VI secretion system (T6SS), and novel antibiotic resistance determinants were identified. A47 exhibited natural transformation with both genomic and plasmid DNA. It was able to form biofilms on different surfaces, to cause hemolysis of sheep and rabbit erythrocytes, and to kill competitor bacteria. Additionally, surface structures with non-uniform length were visualized with scanning electron microscopy and proposed as pili-like structures. Furthermore, the A47 genome revealed the presence of two putative BLUF type photoreceptors, and phenotypic assays confirmed the modulation by light of different virulence traits. Taken together, these results provide insight into the pathobiology of A47, which exhibits multiple virulence factors, natural transformation, and the ability to sense and respond to light, which may contribute to the success of an A47 as a hospital dwelling pathogen.

Integration of Temperature and Blue-Light Sensing in Acinetobacter baumannii Through the BlsA Sensor.

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.

More Than Just Light: Clinical Relevance of Light Perception in the Nosocomial Pathogen Acinetobacter baumannii and Other Members of the Genus Acinetobacter.

A summary of the major findings concerning light modulation in Acinetobacter baumannii, which governs aspects related to the success of this microorganism as a nosocomial pathogen, is presented. Particularly, the evidence shows that light modulates the ability of the bacteria to persist in the environment, its virulence against eukaryotic hosts and even susceptibility to certain antibiotics. The light signal is sensed through different mechanisms, in some cases involving specialized photoreceptors of the BLUF-type, whereas in others, directly by a photosensitizer molecule. We also provide new data concerning the genomic context of BLUF-domain containing proteins within the genus Acinetobacter, as well as further insights into the mechanism of light-mediated reduction in susceptibility to antibiotics. The overall information points toward light being a crucial stimulus in the lifestyle of members of the genus Acinetobacter as well as in other clinically relevant species, such as members of the ESKAPE group, playing therefore an important role in the clinical settings.

White and blue light induce reduction in susceptibility to minocycline and tigecycline in Acinetobacter spp. and other bacteria of clinical importance.

Minocycline (MIN) and tigecycline (TIG) are antibiotics currently used for treatment of multidrug-resistant 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)O2, 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.

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.

Staring at the cold sun: blue light regulation is distributed within the genus Acinetobacter.

We previously showed that the opportunistic nosocomial pathogen Acinetobacter baumannii is able to sense and respond to light via BlsA, a BLUF (Blue-Light-sensing Using FAD)-domain photoreceptor protein. Here, we extend our previous studies showing that light regulation is not restricted to A. baumannii, but rather widespread within the genus Acinetobacter. First, we found that blue light modulates motility and biofilm formation in many species of the genus, including members of the Acinetobacter calcoaceticus-A. baumannii complex. In many of these species blue light acts as a key factor guiding the decision between motility or sessility at 24°C, whereas in A. baumannii, light inhibits both motility and biofilm formation. We also show that light regulation of motility occurred not only at 24°C but also at 37°C in non-A. baumannii species, contrasting the situation of A. baumannii which only shows photoregulation at 24°C. Second, we show that Acinetobacter baylyi (strain ADP1) BLUF-photoreceptors can functionally replace in vivo the A. baumannii 17978 BlsA protein and that the pathways leading to biofilm formation are inversely regulated at 24°C between these two microorganisms. Finally, we found the presence of predicted genes coding BLUF-containing proteins in all Acinetobacter sequenced genomes, even though the copy number is variable among them. Phylogenetic analysis suggests a common origin for all BLUF domains present in members of this genus, and could distinguish well-differentiated clusters that group together BLUF homologs from different species, a situation particularly clear for members of the ACB complex. Despite a role played by these BLUF domain-containing proteins in the photoregulation observed in the members of the genus Acinetobacter is a likely scenario given our findings in A. baumannii and A. baylyi, further research will contribute to confirm this possibility.