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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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

CO 2 and bicarbonate are required for carboxylation reactions, which are essential in most bacteria. To provide the cells with sufficient CO 2 , there exist two dissolved inorganic carbon supply (DICS) systems: the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA). Recently, it has been shown that MpsAB is a bicarbonate transporter that is present not only in photo- and autotrophic bacteria, but also in a diverse range of nonautotrophic microorganisms. Since the two systems rarely coexist in a species but are interchangeable, we investigated what advantages the one system might have over the other. Using the genus Staphylococcus as a model, we deleted the CA gene can in Staphylococcus carnosus and mpsABC genes in Staphylococcus aureus. Deletion of the respective gene in one or the other species led to growth inhibition that could only be reversed by CO 2 supplementation. While the S. carnosus Δ can mutant could be fully complemented with mpsABC , the S. aureus Δ mpsABC mutant was only partially complemented by can , suggesting that MpsAB outperforms CA. Interestingly, we provide evidence that mucus biofilm formation such as that involving polysaccharide intercellular adhesin (PIA) impedes the diffusion of CO 2 into cells, making MpsAB more advantageous in biofilm-producing strains or species. Coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. In conclusion, the distribution of MpsAB or CA in bacteria does not appear to be random as expression of bicarbonate transporters provides an advantage where diffusion of CO 2 is impeded. IMPORTANCE CO 2 and bicarbonate are required for carboxylation reactions in central metabolism and biosynthesis of small molecules in all bacteria. This is achieved by two different systems for dissolved inorganic carbon supply (DICS): these are the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA), but both rarely coexist in a given species. Here, we compared both systems and demonstrate that the distribution of MpsAB and/or CA within the phylum Firmicutes is apparently not random. The bicarbonate transporter MpsAB has an advantage in species where CO 2 diffusion is hampered-for instance, in mucus- and biofilm-forming bacteria. However, coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. Given the clinical relevance of Staphylococcus in the medical environment, such findings contribute to the understanding of bacterial metabolism and thus are crucial for exploration of potential targets for antimicrobials. The knowledge gained here as exemplified by staphylococcal species could be extended to other pathogenic bacteria.

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein-like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat-inducible and appears to play a major role in Lpl1 interaction. Pre-incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1-Hsp90 interaction induces F-actin formation, thus, triggering an endocytosis-like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl-Hsp90 interaction., (© 2019 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

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.

Pseudomonas extremaustralis is a versatile Antarctic bacterium, able to grow under microaerobic and anaerobic conditions and is related to several non-pathogenic Pseudomonads. Here we report on the role of the global anaerobic regulator Anr, in the early steps of P. extremaustralis biofilm development. We found that the anr mutant was reduced in its ability to attach, to form aggregates and to display twitching motility but presented higher swimming motility than the wild type. In addition, microscopy revealed that the wild type biofilm contained more biomass and was thicker, but were less rough than that of the anr mutant. In silico analysis of the P. extremaustralis genome for Anr-like binding sites led to the identification of two biofilm-related genes as potential targets of this regulator. When measured using Quantitative Real Time PCR, we found that the anr mutant expressed lower levels of pilG, which encodes a component of Type IV pili and has been previously implicated in cellular adhesion. Levels of morA, involved in signal transduction and flagella development, were also lower in the mutant. Our data suggest that under low oxygen conditions, such as those encountered in biofilms, Anr differentially regulates aggregation and motility thus affecting the first stages of biofilm formation.

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.

We analyzed the influence of the redox global regulator Anr on the accumulation of poly(3-hydroxybutyrate) (PHB) in Pseudomonas extremaustralis. Anr regulates a set of genes in the aerobic-anaerobic transition including genes involved in nitrate reduction and arginine fermentation. An anr mutant was constructed using PCR-based strategies. The wild-type strain was able to grow in both microaerobic and anaerobic conditions using nitrate as the terminal electron acceptor while the mutant strain was unable to grow under anaerobic conditions. In bioreactor cultures, PHB content in the wild-type strain was higher in microaerobic and anaerobic cultures compared with highly aerated cultures. The mutant strain showed decreased PHB levels in both aerobic and microaerobic conditions compared with the wild-type strain. Inactivation of anr led to decreased expression of phaC and phaR genes as demonstrated in real-time RT-PCR experiments. Associated with the PHB gene region, two putative binding sites for Anr were found that, in line with the phenotype observed in bioreactor cultures, suggest a role of this regulator in PHB biosynthesis., (Copyright © 2010 S. Karger AG, Basel.)