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Antimicrobial compounds play a critical role in combating microbial infections. However, the emergence of antibiotic and antifungal resistance and the scarcity of new antibiotic developments pose a significant threat and demand the discovery of new antimicrobials for both bacterial and fungal pathogens. Our previous work described the first generation ( G1 ) of organoantimony-based compounds that showed antimicrobial activity against several bacterial and fungal pathogens. Here, we present our efforts in modifying these compounds by replacing the tetraphenyl backbone in G1 compounds with a trimethyl group, thereby generating a new series of compounds we refer to as "generation 2", G2 . In addition to the novel backbone structure, we introduced three new anionic chloro-cyanoxime ligand groups, namely 2,4-diCl-PhCO - , 2,6-diCl-PhCO - and 2Cl-PhCO - , which were found to be biologically active in the past. Nine new compounds of SbMe 3 L 2 composition were obtained in high yields and characterized by NMR, IR spectroscopies, thermogravimetric TG/DSC and X-ray single crystal analyses. The antibacterial activity of the cyanoximates was tested against three bacterial ( Pseudomonas aeruginosa PAO1, Escherichia coli S17 and methicillin-resistant Staphylococcus aureus (MRSA) NRS70) and two fungal ( Candida albicans strain SC5314 and Cryptococcus neoformans strain H99) pathogens. Two compounds, SbMe 3 (MCO) 2 and SbMe 3 (2,4-diClPhCO) 2 , were active against bacterial strains and inhibited the growth of PAO1 and MRSA with MICs of 50 and 100 µg/mL, respectively. Three compounds, SbMe 3 (MCO) 2 , SbMe 3 (ECO) 2 and SbMe 3 (TCO) 2 , were active against fungal strains and inhibited either one of or both C. albicans and C. neoformans at MICs of 2.6-66.67 μg/mL. In addition, SbMe 3 (TCO) 2 and SbMe 3 (MCO) 2 were fungicidal at MFC 33.33-66.67 μg/mL. Ultra-thin-layer TEM imaging suggested that SbMe 3 (MCO) 2 targets the integrity of bacterial membranes. Overall, four of the studied G2 series compounds possess antimicrobial activity against a broad range of microbial pathogens, with particular potential against fungal pathogens, which will be explored in further studies.

Ciprofloxacin (CPX) is one of the most employed antibiotics in clinics to date. However, the rise of drug-resistant bacteria is dramatically impairing its efficacy, especially against life-threatening pathogens, such as Pseudomonas aeruginosa . This Gram-negative bacterium is an opportunistic pathogen, often infecting immuno-compromised patients with severe or fatal outcomes. The evidence of the possibility of exploiting Carbonic Anhydrase (CA, EC: 4.2.1.1) enzymes as pharmacological targets along with their role in P. aeruginosa virulence inspired the derivatization of CPX with peculiar CA-inhibiting chemotypes. Thus, a large library of CPX derivatives was synthesized and tested on a panel of bacterial CAs and human isoenzymes I and II. Selected derivatives were evaluated for antibacterial activity, revealing bactericidal and antibiofilm properties for some compounds. Importantly, promising preliminary absorption, distribution, metabolism, and excretion (ADME) properties in vitro were found and no cytotoxicity was detected for some representative compounds when tested in Galleria mellonella larvae.

The human pathogen Pseudomonas aeruginosa ( Pa ) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca 2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca 2+ -binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca 2+ -regulated virulence in P. aeruginosa . Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca 2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca 2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca 2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca 2+ and Fe, and this regulation required a Ca 2+ -dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca 2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca 2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to the host.IMPORTANCE Pseudomonas aeruginosa ( Pa ) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca 2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca 2+ sensor, EfhP, is required for at least 1/3 of the Ca 2+ response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of efhP itself is regulated by Ca 2+ and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca 2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca 2+ and associated regulatory mechanisms will serve in the development of future therapeutics targeting Pa 's dangerous infections., Competing Interests: The authors declare no conflict of interest.

Antimicrobial resistance (AMR) poses a significant worldwide public health crisis that continues to threaten our ability to successfully treat bacterial infections. With the decline in effectiveness of conventional antimicrobial therapies and the lack of new antibiotic pipelines, there is a renewed interest in exploring the potential of metal-based antimicrobial compounds. Antimony-based compounds with a long history of use in medicine have re-emerged as potential antimicrobial agents. We previously synthesized a series of novel organoantimony(V) compounds complexed with cyanoximates with a strong potential of antimicrobial activity against several AMR bacterial and fungal pathogens. Here, five selected compounds were studied for their antibacterial efficacy against three important bacterial pathogens: Pseudomonas aeruginosa , Escherichia coli , and Staphylococcus aureus . Among five tested compounds, SbPh 4 ACO showed antimicrobial activity against all three bacterial strains with the MIC of 50-100 µg/mL. The minimum bactericidal concentration/MIC values were less than or equal to 4 indicating that the effects of SbPh 4 ACO are bactericidal. Moreover, ultra-thin electron microscopy revealed that SbPh 4 ACO treatment caused membrane disruption in all three strains, which was further validated by increased membrane permeability. We also showed that SbPh 4 ACO acted synergistically with the antibiotics, polymyxin B and cefoxitin used to treat AMR strains of P. aeruginosa and S. aureus , respectively, and that at synergistic MIC concentration 12.5 µg/mL, its cytotoxicity against the cell lines, Hela, McCoy, and A549 dropped below the threshold. Overall, the results highlight the antimicrobial potential of novel antimony-based compound, SbPh 4 ACO, and its use as a potentiator of other antibiotics against both Gram-positive and Gram-negative bacterial pathogens., Importance: Antibiotic resistance presents a critical global public health crisis that threatens our ability to combat bacterial infections. In light of the declining efficacy of traditional antibiotics, the use of alternative solutions, such as metal-based antimicrobial compounds, has gained renewed interest. Based on the previously synthesized innovative organoantimony(V) compounds, we selected and further characterized the antibacterial efficacy of five of them against three important Gram-positive and Gram-negative bacterial pathogens. Among these compounds, SbPh 4 ACO showed broad-spectrum bactericidal activity, with membrane-disrupting effects against all three pathogens. Furthermore, we revealed the synergistic potential of SbPh 4 ACO when combined with antibiotics, such as cefoxitin, at concentrations that exert no cytotoxic effects tested on three mammalian cell lines. This study offers the first report on the mechanisms of action of novel antimony-based antimicrobial and presents the therapeutic potential of SbPh 4 ACO in combating both Gram-positive and Gram-negative bacterial pathogens while enhancing the efficacy of existing antibiotics., Competing Interests: The authors declare no conflict of interest.

The human pathogen Pseudomonas aeruginosa poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca 2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca 2+ -binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca 2+ -regulated virulence in P. aeruginosa . Here we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca 2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca 2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ) and several virulence factors, such as production of pyocins. The Ca 2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca 2+ and Fe, and this regulation required Ca 2+ -dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca 2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca 2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to host., Importance: Pseudomonas aeruginosa ( Pa ) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca 2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca 2+ sensor, EfhP, is required for at least 1/3 of the Ca 2+ response, including all the iron uptake mechanisms and production of pyocins. Transcription of efhP itself is regulated by Ca 2+ , Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca 2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca 2+ and associated regulatory mechanisms will serve the development of future therapeutics targeting Pa dangerous infections.

Carbonic anhydrases (CAs) catalyze the reversable hydration of carbon dioxide to bicarbonate placing them into the core of the biochemical carbon cycle. Due to the fundamental importance of their function, they evolved independently into eight classes, three of which have been recently discovered. Most research on CAs has focused on their representatives in eukaryotic organisms, while prokaryotic CAs received significantly less attention. Nevertheless, prokaryotic CAs play a key role in the fundamental ability of the biosphere to acquire CO 2 for photosynthesis and to decompose the organic matter back to CO 2 . They also contribute to a broad spectrum of processes in pathogenic bacteria, enhancing their ability to survive in a host and, therefore, present a promising target for developing antimicrobials. This review focuses on the distribution of CAs among bacterial pathogens and their importance in bacterial virulence and host-pathogen interactions., (Copyright © 2024. Published by Elsevier Inc.)

Calcium signaling is an essential mediator of signal-controlling gene expression in most developmental systems. In addition, calcium has established extracellular functions as a structural component of biogenic minerals found in complex tissues. In bacteria, the formation of calcium carbonate structures is associated with complex colony morphology. Genes promoting the formation of biogenic minerals are essential for proper biofilm development and protection against antimicrobial solutes and toxins. Here we review recent findings on the role of calcium and calcium signaling as emerging regulators of biofilm formation in beneficial bacteria, as well as essential mediators of biofilm formation and virulence in human pathogens. The presented analysis concludes that the new understanding of calcium signaling may help to improve the performance of beneficial strains for sustainable agriculture, microbiome manipulation, and sustainable construction. Unraveling the roles of calcium may also promote the development of novel therapies against biofilm infections that target calcium uptake, calcium sensors, and calcium carbonate deposition., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A series of the eight novel organoantimony(V) cyanoximates of Sb(C 6 H 5 ) 4 L composition was synthesized using the high-yield heterogeneous metathesis reaction between solid AgL (or TlL) and Sb(C 6 H 5 ) 4 Br in CH 3 CN at room temperature. Cyanoximes L were specially selected from a large group of 48 known compounds of this subclass of oximes on the basis of their water solubility and history of prior biological activity. The synthesized compounds are well soluble in organic solvents and were studied using a variety of conventional spectroscopic and physical methods. The crystal structures of all reported organometallic compounds were determined and revealed the formation of the distorted trigonal bipyramidal environment of the Sb atom and monodentate axial binding of acido-ligands via the O atom of the oxime group. The compounds are thermally stable in the solid state and in solution molecular compounds. For the first time, this specially designed series of organoantimony(V) compounds is investigated as potential non-antibiotic antimicrobial agents against three bacterial and two fungal human pathogens known for their increasing antimicrobial resistance. Bacterial pathogens included Gram-negative Escherichia coli and Pseudomonas aeruginosa, and Gram-positive Staphylococcus aureus. Fungal pathogens included Cryptococcus neoformans and Candida albicans. The cyanoximates alone showed no antimicrobial impact, and the incorporation of the SbPh 4 group enabled the antimicrobial effect. Overall, the new antimony compounds showed a strong potential as both broad- and narrow-spectrum antimicrobials against selected bacterial and fundal pathogens and provide insights for further synthetic modifications of the compounds to increase their activities.

Calcium (Ca 2+ ) is well known as a second messenger in eukaryotes, where Ca 2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca 2+ signaling, little is known about the mechanisms of bacterial Ca 2+ signaling. Previously, we have identified a putative Ca 2+ -binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca 2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca 2+ with 13.7 µM affinity, and that mutations at the +X and -Z positions within each or both EF-hand motifs abolished Ca 2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca 2+ -dependent manner, however no such response was detected in the mutated proteins. 15  N-NMR showed Ca 2+ -dependent chemical shifts in EfhP confirming Ca 2+ -binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca 2+ binding abolished Ca 2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca 2+ , which triggers its structural changes required for the Ca 2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca 2+ sensor., (© 2022. The Author(s).)

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening infections. Previously, we showed that elevated calcium (Ca 2+ ) levels increase the production of virulence factors in P. aeruginosa In an effort to characterize the Ca 2+ regulatory network, we identified a Ca2+ - r egulated β- p ropeller protein, CarP, and showed that expression of the encoding gene is controlled by the Ca 2+ -regulated two-component system CarSR. Here, by using a Galleria melonella model, we showed that CarP plays a role in regulating P. aeruginosa virulence. By using transcriptome sequencing (RNA-Seq), reverse transcription (RT)-PCR, quantitative RT-PCR (RT-qPCR), and promoter fusions, we determined that carP is transcribed into at least two transcripts and regulated by several bacterial and host factors. The transcription of carP is elevated in response to Ca 2+ in P. aeruginosa cystic fibrosis isolates and PAO1 laboratory strain. Elevated Fe 2+ also induces carP The simultaneous addition of Ca 2+ and Fe 2+ increased the carP promoter activity synergistically, which requires the presence of CarR. In silico analysis of the intergenic sequence upstream of carP predicted recognition sites of RhlR/LasR, OxyR, and LexA, suggesting regulation by quorum sensing (QS) and oxidative stress. In agreement, the carP promoter was activated in response to stationary-phase PAO1 supernatant and required the presence of elevated Ca 2+ and CarR but remained silent in the triple mutant lacking rhlI, lasI , and pqsA synthases. We also showed that carP transcription is regulated by oxidative stress and that CarP contributes to P. aeruginosa Ca 2+ -dependent H 2 O 2 tolerance. The multifactorial regulation of carP suggests that CarP plays an important role in P. aeruginosa adaptations to host environments. IMPORTANCE P. aeruginosa is a human pathogen causing life-threatening infections. It is particularly notorious for its ability to adapt to diverse environments within the host. Understanding the signals and the signaling pathways enabling P. aeruginosa adaptation is imperative for developing effective therapies to treat infections caused by this organism. One host signal of particular importance is calcium. Previously, we identified a component of the P. aeruginosa calcium-signaling network, CarP, whose expression is induced by elevated levels of calcium. Here, we show that carP plays an important role in P. aeruginosa virulence and is upregulated in P. aeruginosa strains isolated from sputa of patients with cystic fibrosis. We also identified several bacterial and host factors that regulate the transcription of carP Such multifactorial regulation highlights the interconnectedness between regulatory circuits and, together with the pleotropic effect of CarP on virulence, suggests the importance of this protein in P. aeruginosa adaptations to the host., (Copyright © 2021 American Society for Microbiology.)

Pseudomonas aeruginosa infects patients with cystic fibrosis, burns, wounds and implants. Previously, our group showed that elevated Ca 2+ positively regulates the production of several virulence factors in P. aeruginosa , such as biofilm formation, production of pyocyanin and secreted proteases. We have identified a Ca 2+ -regulated β-propeller putative phytase, CarP, which is required for Ca 2+ tolerance, regulation of the intracellular Ca 2+ levels, and plays a role in Ca 2+ regulation of P. aeruginosa virulence. Here, we studied the conservation of carP sequence and its occurrence in diverse phylogenetic groups of bacteria. In silico analysis revealed that carP and its two paralogues PA2017 and PA0319 are primarily present in P. aeruginosa and belong to the core genome of the species. We identified 155 single nucleotide alterations within carP , 42 of which lead to missense mutations with only three that affected the predicted 3D structure of the protein. PCR analyses with carP -specific primers detected P. aeruginosa specifically in 70 clinical and environmental samples. Sequence comparison demonstrated that carP is overall highly conserved in P. aeruginosa isolated from diverse environments. Such evolutionary preservation of carP illustrates its importance for P. aeruginosa adaptations to diverse environments and demonstrates its potential as a biomarker.

Calcium (Ca 2+ ) is a universal signaling ion, whose major informational role shaped the evolution of signaling pathways, enabling cellular communications and responsiveness to both the intracellular and extracellular environments. Elaborate Ca 2+ regulatory networks have been well characterized in eukaryotic cells, where Ca 2+ regulates a number of essential cellular processes, ranging from cell division, transport and motility, to apoptosis and pathogenesis. However, in bacteria, the knowledge on Ca 2+ signaling is still fragmentary. This is complicated by the large variability of environments that bacteria inhabit with diverse levels of Ca 2+ . Yet another complication arises when bacterial pathogens invade a host and become exposed to different levels of Ca 2+ that (1) are tightly regulated by the host, (2) control host defenses including immune responses to bacterial infections, and (3) become impaired during diseases. The invading pathogens evolved to recognize and respond to the host Ca 2+ , triggering the molecular mechanisms of adhesion, biofilm formation, host cellular damage, and host-defense resistance, processes enabling the development of persistent infections. In this review, we discuss: (1) Ca 2+ as a determinant of a host environment for invading bacterial pathogens, (2) the role of Ca 2+ in regulating main events of host colonization and bacterial virulence, and (3) the molecular mechanisms of Ca 2+ signaling in bacterial pathogens.

Calcification of soft tissue leads to serious diseases and has been associated with bacterial chronic infections. However, the origin and the molecular mechanisms of calcification remain unclear. Here we hypothesized that a human pathogen Pseudomonas aeruginosa deposits extracellular calcium, a process requiring carbonic anhydrases (CAs). Transmission electron microscopy confirmed the formation of 0.1-0.2 μm deposits by P. aeruginosa PAO1 growing at 5 mM CaCl 2 , and X-ray elemental analysis confirmed they contain calcium. Quantitative analysis of deposited calcium showed that PAO1 deposits 0.35 and 0.75 mM calcium/mg protein when grown at 5 mM and 10 mM CaCl 2 , correspondingly. Fluorescent microscopy indicated that deposition initiates at the cell surface. We have previously characterized three PAO1 β-class CAs: psCA1, psCA2, and psCA3 that hydrate CO 2 to HCO 3 - , among which psCA1 showed the highest catalytic activity (Lotlikar et. al. 2013). According to immunoblot and RT-qPCR, growth at elevated calcium levels increases the expression of psCA1. Analyses of the deletion mutants lacking one, two or all three psCA genes, determined that psCA1 plays a major role in calcium deposition and contributes to the pathogen's virulence. In-silico modeling of the PAO1 β-class CAs identified four amino acids that differ in psCA1 compared to psCA2, and psCA3 (T59, A61A, A101, and A108), and these differences may play a role in catalytic rate and thus calcium deposition. A series of inhibitors were tested against the recombinant psCA1, among which aminobenzene sulfonamide (ABS) and acetazolamide (AAZ), which inhibited psCA1 catalytic activity with K Is of 19 nM and 37 nM, correspondingly. The addition of ABS and AAZ to growing PAO1 reduced calcium deposition by 41 and 78, respectively. Hence, for the first time, we showed that the β-CA psCA1 in P. aeruginosa contributes to virulence likely by enabling calcium salt deposition, which can be partially controlled by inhibiting its catalytic activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Biofilms are surface-associated microbial communities known for their increased resistance to antimicrobials and host factors. This resistance introduces a critical clinical challenge, particularly in cases associated with implants increasing the predisposition for bacterial infections. Preventing such infections requires the development of novel antimicrobials or compounds that enhance bactericidal effect of currently available antibiotics. We have synthesized and characterized twelve novel silver(I) cyanoximates designated as Ag(ACO), Ag(BCO), Ag(CCO), Ag(ECO), Ag(PiCO), Ag(PICO) (yellow and red polymorphs), Ag(BIHCO), Ag(BIMCO), Ag(BOCO), Ag(BTCO), Ag(MCO) and Ag(PiPCO). The compounds exhibit a remarkable resistance to high intensity visible light, UV radiation and heat and have poor solubility in water. All these compounds can be well incorporated into the light-curable acrylate polymeric composites that are currently used as dental fillers or adhesives of indwelling medical devices. A range of dry weight % from 0.5 to 5.0 of the compounds was tested in this study. To study the potential of these compounds in preventing planktonic and biofilm growth of bacteria, we selected two human pathogens (Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus ) and Gram-positive environmental isolate Bacillus aryabhattai . Both planktonic and biofilm growth was abolished completely in the presence of 0.5% to 5% of the compounds. The most efficient inhibition was shown by Ag(PiCO), Ag(BIHCO) and Ag(BTCO). The inhibition of biofilm growth by Ag(PiCO)-yellow was confirmed by scanning electron microscopy (SEM). Application of Ag(BTCO) and Ag(PiCO)-red in combination with tobramycin, the antibiotic commonly used to treat P. aeruginosa infections, showed a significant synergistic effect. Finally, the inhibitory effect lasted for at least 120 h in P. aeruginosa and 36 h in S. aureus and B. aryabhattai . Overall, several silver(I) cyanoximates complexes efficiently prevent biofilm development of both Gram-negative and Gram-positive bacteria and present a particularly significant potential for applications against P. aeruginosa infections.

Pseudomonas aeruginosa is an opportunistic multidrug resistant pathogen causing severe chronic infections. Our previous studies showed that elevated calcium (Ca 2+ ) enhances production of several virulence factors and plant infectivity of the pathogen. Here we show that Ca 2+ increases resistance of P. aeruginosa PAO1 to tobramycin, antibiotic commonly used to treat Pseudomonas infections. LC-MS/MS-based comparative analysis of the membrane proteomes of P aeruginosa grown at elevated versus not added Ca 2+ , determined that the abundances of two RND (resistance-nodulation-cell division) efflux pumps, MexAB-OprM and MexVW-OprM, were increased in the presence of elevated Ca 2+ . Analysis of twelve transposon mutants with disrupted RND efflux pumps showed that six of them (mexB, muxC, mexY, mexJ, czcB, and mexE) contribute to Ca 2+ -induced tobramycin resistance. Transcriptional analyses by promoter activity and RT-qPCR showed that the expression of mexAB, muxABC, mexXY, mexJK, czcCBA, and mexVW is increased by elevated Ca 2+ . Disruption of mexJ, mexC, mexI, and triA significantly decreased Ca 2+ -induced plant infectivity of the pathogen. Earlier, our group showed that PAO1 maintains intracellular Ca 2+ (Ca 2+ in ) homeostasis, which mediates Ca 2+ regulation of P. aeruginosa virulence, and identified four putative Ca 2+ transporters involved in this process (Guragain et al., 2013). Here we show that three of these transporters (PA2435, PA2092, PA4614) play role in Ca 2+ -induced tobramycin resistance and one of them (PA2435) contributes to Ca 2+ regulation of mexAB-oprM promoter activity. Furthermore, mexJ, czcB, and mexE contribute to the maintenance of Ca 2+ in homeostasis. This provides the first evidence that Ca 2+ in homeostasis mediates Ca 2+ regulation of RND transport systems, which contribute to Ca 2+ -enhanced tobramycin resistance and plant infectivity in P. aeruginosa., (Published by Elsevier Ltd.)

Unlabelled: Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca(2+)) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca(2+) through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca(2+) level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca(2+)-induced virulence factor production and Ca(2+) homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca(2+)] in both planktonic cultures and biofilms. Among the genes induced by CaCl2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR and performed transcriptome analysis of the mutant strain at low and high [Ca(2+)]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca(2+) homeostasis, reducing the ability of P. aeruginosa to export excess Ca(2+). In addition, a mutation in carP had a pleotropic effect in a Ca(2+)-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca(2+) and responding through its regulatory targets that modulate Ca(2+) homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin., Importance: During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca(2+)) concentrations, yet the cells maintain intracellular Ca(2+) at levels that are orders of magnitude less than that of the external environment. In addition, Ca(2+) signals P. aeruginosa to induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca(2+) homeostasis or how Ca(2+) acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca(2+) levels. CarRS modulates Ca(2+) signaling and Ca(2+) homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca(2+) and relays that information for Ca(2+)-dependent cellular processes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The β-class CAs (β-CAs) are expressed in prokaryotes, fungi, plants, and more recently have been isolated in some animals. The β-CA class is divided into two subclasses, termed type I and II, defined by pH catalytic activity profile and active site structural configuration. Type I β-CAs display catalytic activity over a broad pH range (6.5-9.0) with the active site zinc tetrahedrally coordinated by three amino acids and a hydroxide/water. In contrast, type II β-CAs are catalytically active only at a pH 8 and higher where they adopt a functional active site configuration like that of type I. However, below pH 8 they are conformationally self-inactivated by the addition of a fourth amino acid coordinating the zinc and thereby displacing the zinc bound solvent. We have determined the structure of psCA3, a type II β-CA, isolated from Pseudomonas aeruginosa (P. aeruginosa) PAO1 at pH 8.3, in its open active state to a resolution of 1.9 Å. The active site zinc is coordinated by Cys42, His98, Cys101 and a water/hydroxide molecule. P. aeruginosa is a multi-drug resistant bacterium and displays intrinsic resistance to most of the currently used antibiotics; therefore, there is a need for new antibacterial targets. Kinetic data confirm that psCA3 belongs to the type II subclass and that sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. In vivo studies identified that among six tested inhibitors representing sulfonamides, inorganic anions, and small molecules, acetazolamide has the most significant dose-dependent inhibitory effect on P. aeruginosa growth., (Copyright © 2015. Published by Elsevier Ltd.)

Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflammatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 ΔefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H2O2) when cultured at high [Ca2+]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca2+] and no iron. The effect of the ΔefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca2+] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca2+].

Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca(2+)) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca(2+), we characterized Ca(2+) homeostasis in P. aeruginosa PAO1 cells. By using Ca(2+)-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca(2+) ([Ca(2+)]in) is 0.14±0.05μM. In response to external Ca(2+), the [Ca(2+)]in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca(2+) modulated this response. Treatment with inhibitors known to affect Ca(2+) channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca(2+)]in response, suggesting the importance of the corresponding mechanisms in Ca(2+) homeostasis. To identify Ca(2+) transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca(2+)]in homeostasis was monitored for seven Ca(2+)-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca(2+) homeostasis. The lack of PA3920 and vanadate treatment abolished Ca(2+)-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca(2+)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials. Genomic and proteomic studies have identified three genes, pa0102, pa2053 and pa4676, in P. aeruginosa PAO1 encoding three functional β-carbonic anhydrases (β-CAs): psCA1, psCA2 and psCA3, respectively. These β-CAs could serve as novel antimicrobial drug targets for this pathogen. X-ray crystallographic structural studies have been initiated to characterize the structure and function of these proteins. This communication describes the production of two crystal forms (A and B) of β-CA psCA3. Form A diffracted to a resolution of 2.9 Å; it belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and had a calculated Matthews coefficient of 2.23 ų Da⁻¹ assuming four molecules in the crystallographic asymmetric unit. Form B diffracted to a resolution of 3.0 Å; it belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and had a calculated Matthews coefficient of 2.48 ų Da⁻¹ assuming two molecules in the crystallographic asymmetric unit. Preliminary molecular-replacement solutions have been determined with the PHENIX AutoMR wizard and refinement of both crystal forms is currently in progress.

Bacterial β-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO2. They maintain the intracellular balance of CO2/bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative β-CAs that share 28-45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn(2+) and catalyse hydration of CO2 to bicarbonate. PAO102 (psCA1) was 19-26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II β-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO2; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO2. Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism.

Lately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles.

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ∼250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO(2) utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.

The phenylacetyl-CoA (Paa) catabolic pathway and genome-wide gene expression responses to phenylacetate catabolism were studied in the polychlorinated biphenyl (PCB)-degrading strain Burkholderia xenovorans LB400. Microarray and RT-qPCR analyses identified three non-contiguous chromosomal clusters of genes that are predicted to encode a complete Paa pathway that were induced up to 40-fold during growth of LB400 on phenylacetate: paaGHIJKR, paaANEBDF, and paaC. Comparison of the available genome sequences revealed that this organization is unique to Burkholderiaceae. Parallel proteomic studies identified 7 of the 14 predicted Paa proteins, most of which were detected only in phenylacetate-grown cells, but not in benzoate- or succinate-grown cells. Finally, the transcriptomic and proteomic analyses revealed the induction of at least 7 predicted catabolic pathways of aromatic compounds and some aromatic plant products (phenols, mandelate, biphenyl, C(1) compounds, mevalonate, opine, and isoquinoline), as well as an oxidative stress response and a large group of transporters. Most of these genes were not induced during growth on benzoate or biphenyl, suggesting that phenylacetate or a metabolite may act as a signal that triggers multiple physiological processes. Identifying the components of the Paa pathway is important since the pathway appears to contribute to virulence of Burkholderia pathogens.

Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 microM CoCl(2). Ten- to 100-fold less CoCl(2) sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mm phosphate, pH 7.0, 25 degrees C). Divalent metal ions including Co(2+), Cu(2+), Zn(2+), and Ni(2+) stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co(2+) with K(d) of 0.12 +/- 0.06 nM and 110 +/- 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn(2+) (K(d) = 11 +/- 2 nM) and Ni(2+) (K(d) = 49 +/- 17 nM) and did not detectably bind Cu(2+). Substitution of the sole histidine residue did not affect Co(2+) binding. Holo-AnhE had a weak absorption band at 490 nM (epsilon = 9.7 +/- 0.1 m(-1) cm(-1)), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 +/- 0.01 microM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the delta bphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the beta-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.

Rhodococcus sp. RHA1 is a Gram-positive actinomycete capable of metabolizing a wide spectrum of organic compounds whose survival in chemically hostile environments is believed to be in part due to the production of an exocellular polysaccharide (EPS). In order to investigate the functional nature of the EPS, its structure was determined using a combinatory approach including hydrolysis, composition, and methylation, analysis methods, as well as 2D (1)H and (13)C NMR spectroscopy. The EPS was found to be a high-molecular-mass polymer of a repeating tetrasaccharide unit composed of D-glucuronic acid, D-glucose, D-galactose, L-fucose and O-acetyl (1:1:1:1:1), and has the structure:

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on mucous plugs in the lungs of cystic fibrosis (CF) patients, resulting in chronic infections. Pulmonary P. aeruginosa isolates often display a mucoid (alginate-producing) phenotype, whereas non-mucoid strains are generally associated with acute infections. We characterized the cytosolic proteomes of biofilm-associated and planktonic forms of a CF pulmonary isolate, P. aeruginosa FRD1, and a non-mucoid strain, PAO1. Since Ca2+ metabolism is altered in CF pulmonary fluids, we also analysed the effect of Ca2+ on the proteome responses of these strains. Both strains altered the abundances of 40-60% of their proteins in response to biofilm growth and/or [Ca2+]. Differentially expressed proteins clustered into 12 groups, based on their abundance profiles. From these clusters, 146 proteins were identified by using MALDI-TOF/TOF mass spectrometry. Similarities as well as strain-specific differences were observed. Both strains altered the production of proteins involved in iron acquisition, pyocyanin biosynthesis, quinolone signalling and nitrogen metabolism, proteases, and proteins involved in oxidative and general stress responses. Individual proteins from these classes were highly represented in the biofilm proteomes of both strains. Strain-specific differences concerned the proteins within these functional groups, particularly for enzymes involved in iron acquisition and polysaccharide metabolism, and proteases. The results demonstrate that a mucoid CF isolate of P. aeruginosa responds to biofilm-associated growth and [Ca2+] in a fashion similar to strain PAO1, but that strain-specific differences may allow this CF isolate to successfully colonize the pulmonary environment.

Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the DeltabenABCD::kan mutant), the boxC pathway (the DeltaboxABC::kan mutant), and both pathways (the DeltabenABCDDelta boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the DeltabenABCD::kan and DeltabenABCD DeltaboxABC::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the DeltabenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.

Bacteria undergo a variety of physiological changes following a switch from planktonic growth to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular proteomes. Calcium influences these proteome responses, and affects the amount of surface-associated biomass and extracellular matrix material produced by Pseudoalteromonas sp. 1398. Four extracellular proteins, characterized by N-terminal sequencing, showed increased abundances, while one protein, flagellin, showed reduced abundance at higher [Ca(2+)]. Immunoblotting and transmission-electron-microscopy analysis confirmed that higher [Ca(2+)] and surface-associated growth results in the repression of flagella production. Two-dimensional gel electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses demonstrated that Ca(2+) had a greater regulatory influence on Pseudoalteromonas sp. growing in biofilms than on planktonic cultures. Approximately 22 % of the total cytosolic proteins resolved by 2DGE had differing abundances in response to a switch from planktonic growth to surface-associated growth when the cells were cultivated in 1 mM Ca(2+). At higher [Ca(2+)] this number increased to 38 %. Fifteen cellular proteins that were differentially expressed in response to biofilm growth and/or Ca(2+) were analysed by N-terminal sequencing and/or MS/MS. These proteins were identified as factors involved in cellular metabolic functions, putative proteases and transport proteins, although there were several proteins that had not been previously characterized. These results indicate that Ca(2+) causes global changes in matrix material, as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These changes are more pronounced when the bacterium grows in biofilms than when it grows in planktonic culture.

Pseudomonas aeruginosa colonizes the pulmonary tissue of patients with cystic fibrosis (CF), leading to biofilm-associated infections. The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1, calcium addition (1.0 and 10 mM as CaCl(2)) resulted in biofilms that were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.

In gram-negative bacteria, a pathway for aerobic degradation of phenylacetic acid (PAA) that proceeds via phenylacetyl-coenzyme A (CoA) and hydrolytic ring fission plays a central role in the degradation of a range of aromatic compounds. In contrast, the PAA pathway and its role are not well characterized in gram-positive bacteria. A cluster including 13 paa genes encoding enzymes orthologous to those of gram-negative bacteria was identified on the chromosome of Rhodococcus sp. strain RHA1. These genes were transcribed during growth on PAA, with 11 of the genes apparently in an operon yielding a single transcript. Quantitative proteomic analyses revealed that at least 146 proteins were more than twice as abundant in PAA-grown cells of RHA1 than in pyruvate-grown cells. Of these proteins, 29 were identified, including 8 encoded by the paa genes. Knockout mutagenesis indicated that paaN, encoding a putative ring-opening enzyme, was essential for growth on PAA. However, paaF, encoding phenylacetyl-CoA ligase, and paaR, encoding a putative regulator, were not essential. paaN was also essential for growth of RHA1 on phenylacetaldehyde, phenylpyruvate, 4-phenylbutyrate, 2-phenylethanol, 2-phenylethylamine, and l-phenylalanine. In contrast, growth on 3-hydroxyphenylacetate, ethylbenzene, and styrene was unaffected. These results suggest that the range of substrates degraded via the PAA pathway in RHA1 is somewhat limited relative to the range in previously characterized gram-negative bacteria.

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the beta-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the beta-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent beta-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

Aims: The aim of this work was to study the biodegradation of benzyldimethylalkylammonium chloride (BAC) by Aeromonas hydrophila sp. K, an organism isolated from polluted soil and capable of utilizing BAC as sole source of carbon and energy., Methods and Results: High performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) analysis was used to study BAC degradation pathway. It was shown that during BAC biodegradation, formation of benzyldimethylamine, benzylmethylamine, benzylamine, benzaldehyde and benzoic acid occurred. Formation of benzyldimethylamine as the initial metabolite suggested that the cleavage of Calkyl-N bond occurred as the first step of BAC catabolism. Liberation of benzylmethylamine and benzylamine likely resulted from subsequent demethylation reactions, followed by deamination with formation of benzaldehyde. Benzaldehyde was rapidly converted into benzoic acid, which was further degraded., Conclusions: Aer. hydrophila sp. K is able to degrade BAC. A degradation pathway for BAC and related compounds is proposed., Significance and Impact of Study: These findings are significant for understanding biodegradation pathways of benzyl-containing quaternary ammonium compounds.