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Pyomelanin, a polymeric pigment in Pseudomonas, arises mainly from alterations in tyrosine degradation. The chemical structure of pyomelanin remains elusive due to its heterogeneous nature. Here, we report strain-specific differences in pyomelanin structural features across Pseudomonas using PAO1 and PA14 reference strains carrying mutations in hmgA (a gene involved in pyomelanin synthesis), a melanogenic P. aeruginosa clinical isolate (PAM), and a melanogenic P. extremaustralis (PexM). UV spectra showed dual peaks for PAO1 and PA14 mutants and single peaks for PAM and PexM. FTIR phenol : alcohol ratio changes and complex NMR spectra indicated non-linear polymers. UVC radiation survival increased with pyomelanin addition, correlating with pigment absorption attenuation. P. extremaustralis UVC survival varied with melanin source, with PAO1 pyomelanin being the most protective. These findings delineate structure-based pyomelanin subgroups, having distinct physiological effects.

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Bacteria are exposed to reactive oxygen and nitrogen species that provoke oxidative and nitrosative stress which can lead to macromolecule damage. Coping with stress conditions involves the adjustment of cellular responses, which helps to address metabolic challenges. In this study, we performed a global transcriptomic analysis of the response of Pseudomonas extremaustralis to nitrosative stress, induced by S-nitrosoglutathione (GSNO), a nitric oxide donor, under microaerobic conditions. The analysis revealed the upregulation of genes associated with inositol catabolism; a compound widely distributed in nature whose metabolism in bacteria has aroused interest. The RNAseq data also showed heightened expression of genes involved in essential cellular processes like transcription, translation, amino acid transport and biosynthesis, as well as in stress resistance including iron-dependent superoxide dismutase, alkyl hydroperoxide reductase, thioredoxin, and glutathione S-transferase in response to GSNO. Furthermore, GSNO exposure differentially affected the expression of genes encoding nitrosylation target proteins, encompassing metalloproteins and proteins with free cysteine and /or tyrosine residues. Notably, genes associated with iron metabolism, such as pyoverdine synthesis and iron transporter genes, showed activation in the presence of GSNO, likely as response to enhanced protein turnover. Physiological assays demonstrated that P. extremaustralis can utilize inositol proficiently under both aerobic and microaerobic conditions, achieving growth comparable to glucose-supplemented cultures. Moreover, supplementing the culture medium with inositol enhances the stress tolerance of P. extremaustralis against combined oxidative-nitrosative stress. Concordant with the heightened expression of pyoverdine genes under nitrosative stress, elevated pyoverdine production was observed when myo-inositol was added to the culture medium. These findings highlight the influence of nitrosative stress on proteins susceptible to nitrosylation and iron metabolism. Furthermore, the activation of myo-inositol catabolism emerges as a protective mechanism against nitrosative stress, shedding light on this pathway in bacterial systems, and holding significance in the adaptation to unfavorable conditions.

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BACKGROUND: Guanine crystals are organic biogenic crystals found in many organisms. Due to their exceptionally high refractive index, they contribute to structural color and are responsible for the reflective effect in the skin and visual organs in animals such as fish, reptiles, and spiders. Occurrence of these crystals in animals has been known for many years, and they have also been observed in eukaryotic microorganisms, but not in prokaryotes. RESULTS: In this work, we report the discovery of extracellular crystals formed by bacteria and reveal that they are composed of guanine monohydrate. This composition differs from that of biogenic guanine crystals found in other organisms, mostly composed of ß anhydrous guanine. We demonstrate the formation of these crystals by Aeromonas and other bacteria and investigate the metabolic traits related to their synthesis. In all cases studied, the presence of the bacterial guanine crystals correlates with the absence of guanine deaminase, which could lead to guanine accumulation providing the substrate for crystal formation. CONCLUSIONS: Our finding of the hitherto unknown guanine crystal occurrence in prokaryotes extends the range of organisms that produce these crystals to a new domain of life. Bacteria constitute a novel and more accessible model to study the process of guanine crystal formation and assembly. This discovery opens countless chemical and biological questions, including those about the functional and adaptive significance of their production in these microorganisms. It also paves the road for the development of simple and convenient processes to obtain biogenic guanine crystals for diverse applications.

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Environmental changes and human activities can alter the structure and diversity of aquatic microbial communities. In this work, we analyzed the bacterial community dynamics of an urban stream to understand how these factors affect the composition of river microbial communities. Samples were taken from a stream situated in Buenos Aires, Argentina, which flows through residential, peri-urban horticultural, and industrial areas. For sampling, two stations were selected: one influenced by a series of industrial waste treatment plants and horticultural farms (PL), and the other influenced by residential areas (R). Microbial communities were analyzed by sequence analysis of 16S rRNA gene amplicons along an annual cycle. PL samples showed high nutrient content compared with R samples. The diversity and richness of the R site were more affected by seasonality than those of the PL site. At the amplicon sequence variants level, beta diversity analysis showed a differentiation between cool-season (fall and winter) and warm-season (spring and summer) samples, as well as between PL and R sites. This demonstrated that there is spatial and temporal heterogeneity in the composition of the bacterial community, which should be considered if a bioremediation strategy is applied. The taxonomic composition analysis also revealed a differential seasonal cycle of phototrophs and chemoheterotrophs between the sampling sites, as well as different taxa associated with each sampling site. This analysis, combined with a comparative analysis of global rivers, allowed us to determine the genera Arcobacter, Simplicispira, Vogesella, and Sphingomonas as potential bioindicators of anthropogenic disturbance.

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Pseudomonas species are metabolically versatile bacteria able to exploit a wide range of ecological niches. Different Pseudomonas species can grow as free-living cells, biofilms, or associated with plants or animals, including humans, and their ecological success partially lies in their ability to grow and adapt to different temperatures. These bacteria are relevant for human activities, due to their clinical importance and their biotechnological potential for different applications such as bioremediation and the production of biopolymers, surfactants, secondary metabolites, and enzymes. In agriculture, some of them can act as plant growth promoters and are thus used as inoculants, whereas others, like P. syringae pathovars, can cause disease in commercial crops. This review aims to provide an overview of the temperature-response mechanisms in Pseudomonas species, looking for novel features or strategies based on techniques such as transcriptomics and proteomics. We focused on temperature-dependent traits mainly associated with virulence, host colonization, survival, and production of secondary metabolites. We analyzed human, animal, and plant pathogens and plant growth-promoting Pseudomonas species, including P. aeruginosa, P. plecoglossicida, several P. syringae pathovars, and P. protegens. Our aim was to provide a comprehensive view of the relevance of temperature-response traits in human and animal health and agricultural applications. Our analysis showed that features relevant to the bacterial-host interaction are adjusted to the environmental or host temperature regardless of the optimal growth temperature in the laboratory, and thus contribute to improving bacterial fitness. KEY POINTS: • In Pseudomonas species, temperature impacts the bacterial-host interaction. • Interaction traits are expressed at temperatures different from the optimal reported. • The bacterial-host interaction could be affected by climate change.

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Bacterial small non-coding RNAs (sRNAs) play key roles as genetic regulators, mediating in the adaptability to changing environmental conditions and stress responses. In this work, we analysed putative sRNAs identified by RNA-seq experiments in different aeration conditions in the extremophile bacterium P. extremaustralis. These analyses allowed the identification of 177 putative sRNAs under aerobiosis (A), microaerobiosis (M) and microaerobiosis after H2 O2 exposure (m-OS). The size and transcription profile of eight sRNAs with differential expression were verified by Northern blot. sRNA40, with unknown function but conserved in other Pseudomonas species, was selected to perform overexpression experiments followed by RNA-seq analysis. The overexpression of sRNA40 in P. extremaustralis resulted in significant expression changes of 19 genes with 14 differentially upregulated and five downregulated. Among the upregulated genes, eight transcripts corresponded to components of secretion systems, such as gspH, gspK, and gspM, belonging to the Type II secretion system, and rspO and rspP from Type III secretion system. Our results showed a novel sRNA which expression was triggered by low oxygen levels, and whose overexpression was associated with upregulation of selected components of protein secretion systems.

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The original version of this article contains error for some of the authors corrections were not included during correction stage especially for Table 1.

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The production of black pigments in bacteria was discovered more than a century ago and related to tyrosine metabolism. However, their diverse biological roles and the control of melanin synthesis in different bacteria have only recently been investigated. The broad distribution of these pigments suggests that they have an important role in a variety of organisms. Melanins protect microorganisms from many environmental stress conditions, ranging from ultraviolet radiation and toxic heavy metals to oxidative stress. Melanins can also affect bacterial interactions with other organisms and are important in pathogenesis and survival in many environments. Bacteria produce several types of melanin through dedicated pathways or as a result of enzymatic imbalances in altered metabolic routes. The control of the melanin synthesis in bacteria involves metabolic and transcriptional regulation, but many aspects remain still largely unknown. The diverse properties of melanins have spurred a large number of applications, and recent efforts have been done to produce the pigment at biotechnologically relevant scales.

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Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance, able to grow under cold conditions. It is capable to produce polyhydroxyalkanoates (PHAs) mainly as polyhydroxybutyrate (PHB) and, to a lesser extent, medium-chain length polyhydroxyalkanoates (mclPHAs). In this work, we analyzed the role of PHAs and cold adaptation in the survival of P. extremaustralis after lethal UVA exposure. P. extremaustralis presented higher radiation resistance under polymer accumulation conditions. This result was also observed in the derivative mutant strain PHA-, deficient for mclPHAs production. On the contrary, the PHB- derivative mutant, deficient for PHB production, showed high sensitivity to UVA exposure. Complementation of the PHB- strain restored the wild-type resistance level, indicating that the UVA-sensitive phenotype is due to the lack of PHB. All strains exhibited high sensitivity to radiation when cultured under PHAs non-accumulation conditions. A slight decrease in PHB content was observed after UVA exposure in association with increased survival. The scattering of UVA radiation by intracellular PHAs granules could also result in bacterial cell protection. In addition, cold conditions improved UVA tolerance, probably depending on PHB mobilization. Results showed that PHB accumulation is crucial in the resistance to UVA in P. extremaustralis. Mechanisms involved probably entail depolymerization and light scattering acting as a screen, both conferring protection against oxidative stress.

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Reactive oxygen species and nitrogen species (ROS and RNS), produced in a wide range of physiological process even under low oxygen availability, are among the main stressors found in the environment. Strategies developed to combat them constitute key features in bacterial adaptability and survival. Pseudomonas extremaustralis is a metabolic versatile and stress resistant Antarctic bacterium, able to grow under different oxygen conditions. The present work explores the effect of oxidative stress under low oxygen conditions in P. extremaustralis, by combining RNA deep sequencing analysis and physiological studies. Cells grown under microaerobiosis exhibited more oxidative damage in macromolecules and lower survival rates than under aerobiosis. RNA-seq analysis showed an up-regulation of genes related with oxidative stress response, flagella, chemotaxis and biofilm formation while chaperones and cytochromes were down-regulated. Microaerobic cultures exposed to H2O2 also displayed a hyper-flagellated phenotype coupled with a high motility behavior. Moreover, cells that were subjected to oxidative stress presented increased biofilm formation. Altogether, our results suggest that a higher motile behavior and augmented capacity to form biofilm structures could work in addition to well-known antioxidant enzymes and non-enzymatic ROS scavenging mechanisms to cope with oxidative stress at low oxygen tensions.

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A comparative genome analysis of the global anaerobic regulator Anr regulon in five species of Pseudomonas with different life style was performed. Expression of this regulator was detected in all analyzed Pseudomonas. The predicted Anr regulon (pan-regulon) consisted of 253 genes. However, only 11 Anr-boxes located upstream of qor/hemF, hemN, cioA/PA3931, azu, rpsL, gltP, orthologous to PA2867, cspD, tyrZ, slyD and oprG, were common to all species. Whole genome in silico prediction of metabolic pathways identified genes belonging to heme biosynthesis, cytochromes and Entner-Doudoroff pathway as members of Anr regulon in all strains. Extending genome analysis to 28 Pseudomonas spp. spanning all phylogenetic groups showed Anr-boxes conservation in genes related to these functions. When present, genes related to anaerobic metabolism were predicted to hold Anr-boxes. Focused on the genomes of eight P. aeruginosa isolates of diverse origins, we observed a conserved regulon, sharing nearly 80% of the genes, indicating its key role in this opportunistic pathogen. The results suggest that the core Anr regulon comprises genes involved in central metabolism and aerobic electron transport chain, whereas those genes related to anaerobic metabolism and other functions constitute the accessory Anr-regulon, thereby differentially contributing to the ecological fitness of each Pseudomonas species.

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Environments co-contaminated with heavy metals and hydrocarbons have become an important problem worldwide, especially due to the effect of metals on hydrocarbon degrading microorganisms. Pseudomonas extremaustralis, a bacterium isolated from a pristine pond in Antarctica, showed high capabilities to cope with environmental stress and a very versatile metabolism that includes alkane degradation under microaerobic conditions. In this work, we analyzed P. extremaustralis' capability to resist high copper concentrations and the effect of copper presence in diesel biodegradation. We observed that P. extremaustralis resisted up to 4 mM CuSO4 in a rich medium such as LB. This copper resistance is sustained by the presence of the cus and cop operons together with other efflux systems and porins located in a single region in P. extremaustralis genome. When copper was present, diesel degradation was negatively affected, even though copper enhanced bacterial attachment to hydrocarbons. However, when a small amount of glucose (0.05% w/v) was added, the presence of CuSO4 enhanced alkane degradation. In addition, atomic force microscopy analysis showed that the presence of glucose decreased the negative effects produced by copper and diesel on the cell envelopes.

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The environmental strain Aeromonas salmonicida subsp. pectinolytica 34melT produces abundant melanin through the homogentisate pathway in several culture media, but unexpectedly not when grown in a medium containing glycerol. Using this observation as a starting point, this study investigated the underlying causes of the inhibition of melanin synthesis by glycerol, to shed light on factors that affect melanin production in this microorganism. The effect of different carbon sources on melanin formation was related to the degree of oxidation of their C atoms, as the more reduced substrates delayed melanization more than the more oxidized ones, although only glycerol completely abolished melanin production. Glyphosate, an inhibitor of aromatic amino acid synthesis, did not affect melanization, while bicyclopyrone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (Hpd), the enzyme responsible for the synthesis of homogentisate, prevented melanin synthesis. These results showed that melanin production in 34melT depends on the degradation of aromatic amino acids from the growth medium and not on de novo aromatic amino acid synthesis. The presence of glycerol changed the secreted protein profile, but none of the proteins affected could be directly connected with melanin synthesis or transport. Transcription analysis of hpd, encoding the key enzyme for melanin synthesis, showed a clear inhibition caused by glycerol. The results obtained in this work indicate that a significant decrease in the transcription of hpd, together with a more reduced intracellular state, would lead to the abolishment of melanin synthesis observed. The effect of glycerol on melanization can thus be attributed to a combination of metabolic and regulatory effects.

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The genus Coprothermobacter (initially named Thermobacteroides) is currently placed within the phylum Firmicutes. Early 16S rRNA gene based phylogenetic studies pointed out the great differences between Coprothermobacter and other members of the Firmicutes, revealing that it constitutes a new deep branching lineage. Over the years, several studies based on 16S rRNA gene and whole genome sequences have indicated that Coprothermobacter is very distant phylogenetically to all other bacteria, supporting its placement in a distinct deeply rooted novel phylum. In view of this, we propose its allocation to the new family Coprothermobacteraceae within the novel order Coprothermobacterales, the new class Coprothermobacteria, and the new phylum Coprothermobacterota, and an emended description of the family Thermodesulfobiaceae.

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It is well known that cold environments are predominant over the Earth and there are a great number of reports analyzing bacterial adaptations to cold. Most of these works are focused on characteristics traditionally involved in cold adaptation, such as the structural adjustment of enzymes, maintenance of membrane fluidity, expression of cold shock proteins and presence of compatible solutes. Recent works based mainly on novel "omic" technologies have presented evidence of the presence of other important features to thrive in cold. In this work, we analyze cold-adapted bacteria, looking for strategies involving novel features, and/or activation of non-classical metabolisms for a cold lifestyle. Metabolic traits related to energy generation, compounds and mechanisms involved in stress resistance and cold adaptation, as well as characteristics of the cell envelope, are analyzed in heterotrophic cold-adapted bacteria. In addition, metagenomic, metatranscriptomic and metaproteomic data are used to detect key functions in bacterial communities inhabiting cold environments.

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Psychrotroph microorganisms have developed cellular mechanisms to cope with cold stress. Cell envelopes are key components for bacterial survival. Outer membrane is a constituent of Gram negative bacterial envelopes, consisting of several components, such as lipopolysaccharides (LPS). In this work we investigated the relevance of envelope characteristics for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis by analyzing a mini Tn5 wapH mutant strain, encoding a core LPS glycosyltransferase. Our results showed that wapH strain is impaired to grow under low temperature but not for cold survival. The mutation in wapH, provoked a strong aggregative phenotype and modifications of envelope nanomechanical properties such as lower flexibility and higher turgor pressure, cell permeability and surface area to volume ratio (S/V). Changes in these characteristics were also observed in the wild type strain grown at different temperatures, showing higher cell flexibility but lower turgor pressure under cold conditions. Cold shock experiments indicated that an acclimation period in the wild type is necessary for cell flexibility and S/V ratio adjustments. Alteration in cell-cell interaction capabilities was observed in wapH strain. Mixed cells of wild type and wapH strains, as well as those of the wild type strain grown at different temperatures, showed a mosaic pattern of aggregation. These results indicate that wapH mutation provoked marked envelope alterations showing that LPS core conservation appears as a novel essential feature for active growth under cold conditions.

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Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13-C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments.

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Solar UVA radiation is one of the main environmental stress factors for Pseudomonas aeruginosa. Exposure to high UVA doses produces lethal effects by the action of the reactive oxygen species (ROS) it generates. P. aeruginosa has several enzymes, including KatA and KatB catalases, which provide detoxification of ROS. We have previously demonstrated that KatA is essential in defending P. aeruginosa against high UVA doses. In order to analyse the mechanisms involved in the adaptation of this micro-organism to UVA, we investigated the effect of exposure to low UVA doses on KatA and KatB activities, and the physiological consequences. Exposure to UVA induced total catalase activity; assays with non-denaturing polyacrylamide gels showed that both KatA and KatB activities were increased by radiation. This regulation occurred at the transcriptional level and depended, at least partly, on the increase in H2O2 levels. We demonstrated that exposure to low UVA produced a protective effect against subsequent lethal doses of UVA, sodium hypochlorite and H2O2. Protection against lethal UVA depends on katA, whilst protection against sodium hypochlorite depends on katB, demonstrating that different mechanisms are involved in the defence against these oxidative agents, although both genes can be involved in the global cellular response. Conversely, protection against lethal doses of H2O2 could depend on induction of both genes and/or (an)other defensive factor(s). A better understanding of the adaptive response of P. aeruginosa to UVA is relevant from an ecological standpoint and for improving disinfection strategies that employ UVA or solar irradiation.

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Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures. Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deep-sequencing technology of early exponential cultures of P. extremaustralis growing in LB (Luria Broth) supplemented with sodium octanoate to favor PHA accumulation at 8°C and 30°C. We found that genes involved in primary metabolism, including tricarboxylic acid cycle (TCA) related genes, as well as cytochromes and amino acid metabolism coding genes, were repressed at low temperature. Among up-regulated genes, those coding for transcriptional regulatory and signal transduction proteins were over-represented at cold conditions. Remarkably, we found that genes involved in ethanol oxidation, exaA, exaB and exaC, encoding a pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the cytochrome c550 and an aldehyde dehydrogenase respectively, were up-regulated. Along with RNA-seq experiments, analysis of mutant strains for pqqB (PQQ biosynthesis protein B) and exaA were carried out. We found that the exaA and pqqB genes are essential for growth under low temperature in LB supplemented with sodium octanoate. Additionally, p-rosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures grown at 8°C support the conclusion that this pathway is important under cold conditions. The obtained results have led to the identification of novel components involved in cold adaptation mechanisms in this bacterium, suggesting for the first time a role of the ethanol oxidation pathway for bacterial growth at low temperatures.

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Bacterial polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in central metabolism, as they act as dynamic reservoirs of carbon and reducing equivalents. These polymers have a number of technical applications since they exhibit thermoplastic and elastomeric properties, making them attractive as a replacement of oil-derived materials. PHAs are accumulated under conditions of nutritional imbalance (usually an excess of carbon source with respect to a limiting nutrient, such as nitrogen or phosphorus). The cycle of PHA synthesis and degradation has been recognized as an important physiological feature when these biochemical pathways were originally described, yet its role in bacterial processes as diverse as global regulation and cell survival is just starting to be appreciated in full. In the present revision, the complex regulation of PHA synthesis and degradation at the transcriptional, translational, and metabolic levels are explored by analyzing examples in natural producer bacteria, such as Pseudomonas species, as well as in recombinant Escherichia coli strains. The ecological role of PHAs, together with the interrelations with other polymers and extracellular substances, is also discussed, along with their importance in cell survival, resistance to several types of environmental stress, and planktonic-versus-biofilm lifestyle. Finally, bioremediation and plant growth promotion are presented as examples of environmental applications in which PHA accumulation has successfully been exploited.

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