Guanine crystal formation by bacteria.

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.

Spatial and Seasonal Variations in the Bacterial Community of an Anthropogenic Impacted Urban Stream.

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.

Insights into the temperature responses of Pseudomonas species in beneficial and pathogenic host interactions.

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.

Response to lethal UVA radiation in the Antarctic bacterium Pseudomonas extremaustralis: polyhydroxybutyrate and cold adaptation as protective factors.

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.

Correction to: Melanin biosynthesis in bacteria, regulation and production perspectives.

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

Melanin biosynthesis in bacteria, regulation and production perspectives.

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.

Effect of copper on diesel degradation in Pseudomonas extremaustralis.

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.

Oxidative stress under low oxygen conditions triggers hyperflagellation and motility in the Antarctic bacterium Pseudomonas extremaustralis.

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.

Glycerol inhibition of melanin biosynthesis in the environmental Aeromonas salmonicida 34melT.

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.

Proposal for a new classification of a deep branching bacterial phylogenetic lineage: transfer of Coprothermobacter proteolyticus and Coprothermobacter platensis to Coprothermobacteraceae fam. nov., within Coprothermobacterales ord. nov., Coprothermobacteria classis nov. and Coprothermobacterota phyl. nov. and emended description of the family Thermodesulfobiaceae.

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.

Microaerophilic alkane degradation in Pseudomonas extremaustralis: a transcriptomic and physiological approach.

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.

Arbuscular Mycorrhizal Fungal Association in Genetically Modified Drought-Tolerant Corn.

The commercial use of genetically modified (GM) plants has significantly increased worldwide. The interactions between GM plants and arbuscular mycorrhizal (AM) fungi are of considerable importance given the agricultural and ecological role of AM and the lack of knowledge regarding potential effects of drought-tolerant GM corn ( L.) on AM fungal symbiosis. This work studied AM fungal colonization in five corn lines growing under two different irrigation regimes (30 and 100% of soil field capacity [SFC]). Four of the lines were GM corn, and two of these were drought tolerant. The experiment was conducted for 60 d in a growth chamber under constant irrigation, after which mycorrhization, corn biomass, and days to plant senescence (DTS) were evaluated. Arbuscular mycorrhizal fungal species of the order were predominant in the soil inocula. At the end of the experiment, all plants showed AM colonization. Mycorrhization was higher at 30% SFC than at 100% SFC. Within the same corn line, the AM fungi produced more vesicles in plant roots under drought stress. Among treatments, DTS varied significantly, and drought-tolerant GM corn lines survived longer than the wild-type corn when maintained at 100% SFC. Corn biomass did not vary among treatments, and no correlations were found between DTS or biomass and mycorrhization. We conclude that overexpression of the gene in corn plants under the experimental conditions of this study did not affect AM fungal infectivity and improved the tolerance of the corn to drought stress.

Exposure to low UVA doses increases KatA and KatB catalase activities, and confers cross-protection against subsequent oxidative injuries in Pseudomonas aeruginosa.

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.

Living in an Extremely Polluted Environment: Clues from the Genome of Melanin-Producing Aeromonas salmonicida subsp. pectinolytica 34melT.

Aeromonas salmonicida subsp. pectinolytica 34mel(T) can be considered an extremophile due to the characteristics of the heavily polluted river from which it was isolated. While four subspecies of A. salmonicida are known fish pathogens, 34mel(T) belongs to the only subspecies isolated solely from the environment. Genome analysis revealed a high metabolic versatility, the capability to cope with diverse stress agents, and the lack of several virulence factors found in pathogenic Aeromonas. The most relevant phenotypic characteristics of 34mel(T) are pectin degradation, a distinctive trait of A. salmonicida subsp. pectinolytica, and melanin production. Genes coding for three pectate lyases were detected in a cluster, unique to this microorganism, that contains all genes needed for pectin degradation. Melanin synthesis in 34mel(T) is hypothesized to occur through the homogentisate pathway, as no tyrosinases or laccases were detected and the homogentisate 1,2-dioxygenase gene is inactivated by a transposon insertion, leading to the accumulation of the melanin precursor homogentisate. Comparative genome analysis of other melanogenic Aeromonas strains revealed that this gene was inactivated by transposon insertions or point mutations, indicating that melanin biosynthesis in Aeromonas occurs through the homogentisate pathway. Horizontal gene transfer could have contributed to the adaptation of 34mel(T) to a highly polluted environment, as 13 genomic islands were identified in its genome, some of them containing genes coding for fitness-related traits. Heavy metal resistance genes were also found, along with others associated with oxidative and nitrosative stresses. These characteristics, together with melanin production and the ability to use different substrates, may explain the ability of this microorganism to live in an extremely polluted environment.

Polyhydroxyalkanoates: Much More than Biodegradable Plastics.

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.

Genome sequence analysis of Pseudomonas extremaustralis provides new insights into environmental adaptability and extreme conditions resistance.

The genome of the Antarctic bacterium Pseudomonas extremaustralis was analyzed searching for genes involved in environmental adaptability focusing on anaerobic metabolism, osmoregulation, cold adaptation, exopolysaccharide production and degradation of complex compounds. Experimental evidences demonstrated the functionality of several of these pathways, including arginine and pyruvate fermentation, alginate production and growth under cold conditions. Phylogenetic analysis along with genomic island prediction allowed the detection of genes with probable foreign origin such as those coding for acetate kinase, osmotic resistance and colanic acid biosynthesis. These findings suggest that in P. extremaustralis the horizontal transfer events and/or gene redundancy could play a key role in the survival under unfavorable conditions. Comparative genome analysis of these traits in other representative Pseudomonas species highlighted several similarities and differences with this extremophile bacterium.

Polyhydroxyalkanoate synthesis affects biosurfactant production and cell attachment to hydrocarbons in Pseudomonas sp. KA-08.

Stressful conditions prevailing in hydrocarbon-contaminated sites influence the diversity, distribution, and activities of microorganisms. Oil bioremediation agents should develop special characteristics to cope with these environments like surfactant production and cellular affinity to hydrocarbons. Additionally, polyhydroxyalkanoate (PHA) accumulation was proven to improve tolerance to stressful conditions. Pseudomonas sp. KA-08 was isolated from a chronic oil-contaminated environment, it is highly tolerant to xylene, and it is able to accumulate PHA and to produce surfactant compounds that lower the water surface tension (ST) as well as bioemulsifiers. In this work, we studied the effect of the capability to accumulate PHAs on biosurfactant production and microbial attachment to hydrocarbons (MATH). Our results showed that PHA synthesis capability has a favorable effect in the production of compounds which affect the ST but not on the production of bioemulsifiers. On the other hand, PHA accumulation affects cellular affinity to xylene. MATH analysis showed that a PHA-negative mutant increased its affinity to xylene compared with the wild-type strain. This result was also observed in Pseudomonas putida GPp104 (a PHA(-) mutant), suggesting that this effect could be generalized to other Pseudomonas strains.

Nitrosative stress under microaerobic conditions triggers inositol metabolism in Pseudomonas extremaustralis.

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.

Anr, the anaerobic global regulator, modulates the redox state and oxidative stress resistance in Pseudomonas extremaustralis.

The role of Anr in oxidative stress resistance was investigated in Pseudomonas extremaustralis, a polyhydroxybutyrate-producing Antarctic bacterium. The absence of Anr caused increased sensitivity to hydrogen peroxide under low oxygen tension. This phenomenon was associated with a decrease in the redox ratio, higher oxygen consumption and higher reactive oxygen species production. Physiological responses of the mutant to the oxidized state included an increase in NADP(H) content, catalase activity and exopolysaccharide production. The wild-type strain showed a sharp decrease in the reduced thiol pool when exposed to hydrogen peroxide, not observed in the mutant strain. In silico analysis of the genome sequence of P. extremaustralis revealed putative Anr binding sites upstream from genes related to oxidative stress. Genes encoding several chaperones and cold shock proteins, a glutathione synthase, a sulfate transporter and a thiol peroxidase were identified as potential targets for Anr regulation. Our results suggest a novel role for Anr in oxidative stress resistance and in redox balance maintenance under conditions of restricted oxygen supply.

Genome sequence of the polyhydroxybutyrate producer Pseudomonas extremaustralis, a highly stress-resistant Antarctic bacterium.

Pseudomonas extremaustralis 14-3b presents genes involved in the synthesis of different polyhydroxyalkanoates, in tolerance and degradation of pollutants, and in microaerobic metabolism. Several genomic islands were detected. Genetic machinery could contribute to the adaptability to stressful conditions. This is the first genome sequence reported from a Pseudomonas isolated from cold environments.