Bacterial consortium proteomics under 4‐chlorosalicylate carbon‐limiting conditions

In this study, the stable consortium composed by Pseudomonas reinekei strain MT1 and Achromobacter xylosoxidans strain MT3 (cell numbers in proportion 9:1) was under investigation to reveal bacterial interactions that take place under severe nutrient‐limiting conditions. The analysis of steady states in continuous cultures was carried out at the proteome, metabolic profile, and population dynamic levels. Carbon‐limiting studies showed a higher metabolic versatility in the community through upregulation of parallel catabolic enzymes (salicylate 5‐hydroxylase and 17‐fold on 2‐keto‐4‐pentenoate hydratase) indicating a possible alternative carbon routing in the upper degradation pathway highlighting the effect of minor proportions of strain MT3 over the major consortia component strain MT1 with a significant change in the expression levels of the enzymes of the mainly induced biodegradation pathway such as salicylate 1‐hydroxylase and catechol 1,2‐dioxygenase together with important changes in the outer membrane composition of P. reinekei MT1 under different culture conditions. The study has demonstrated the importance of the outer membrane as a sensing/response protective barrier caused by interspecies interactions highlighting the role of the major outer membrane proteins OprF and porin D in P. reinekei sp. MT1 under the culture conditions tested.     

Kinetics of carbon sharing in a bacterial consortium revealed by combining stable isotope probing with fluorescence‐activated cell sorting

Aims: To determine the kinetics of substrate fluxes in a microbial community in order to elucidate the roles of the community members. Methods and Results: The kinetics of substrate sharing in a bacterial consortium were measured by a new analytical approach combining immunostaining, stable isotope probing and fluorescence‐activated cell sorting (FACS). The bacterial consortium, consisting of four strains and growing on 4‐chlorosalicylate (4‐CS), was pulse‐dosed with the degradation intermediate [U‐¹³C]‐4‐chlorocatechol (4‐CC). Cells were stained with strain‐specific antibodies sorted by FACS and the ¹³C‐incorporation into fatty acids of the two most abundant members of the community was determined by isotope ratio mass spectrometry. From the two most abundant strains, the primary degrader Pseudomonas reinekei MT1 incorporated the labelled substrate faster than strain Achromobacter spanius MT3 but the maximal incorporation in strain MT3 was almost three times higher than in MT1. Conclusions: It has been reported that strain MT1 produces 4‐CC as an intermediate but has a lower LD₅₀ for it than strain MT3; therefore, MT3 still degrades 4‐CC when the concentrations of 4‐CC are already too toxic, even lethal, for MT1. By degrading 4‐CC, produced by MT1, MT3 protects the entire community against this toxin. The higher affinity but lower tolerance of strain MT1 for 4‐chlorocatechol compared to strain MT3 explains the complementary function these two strains have in the consortium adding exceptional stability to the entire community. Significance and Impact of the Study: The novel approach can reveal carbon fluxes in microbial communities generating quantitative data for systems biology of the microbial community.     

In this issue: Proteomics 8/2009

In this issue of Proteomics you will find the following highlighted articles: Are you sick or are you just getting old: Does it matter? We all joke about the hazards of aging: various systems that break down, some you didn't even know you had. But then there's the alternative of not aging. Hmmm. Now what if aging were a disease? If so, it is worse than any cold I've had. Zürbig et al. have found that the proteome of the aging kidney has many markers in common with chronic kidney disease. The degree of match among small peptide markers ranged from 4% to 22% for IgA nephropathy to diabetic nephropathy, respectively. From these data they developed an age estimating scale that revealed some individuals had kidneys apparently “older” than their bodies. If these findings hold up, they could offer new approaches to diagnosis and therapy of chronic kidney diseases. Zürbig, P. et al., Proteomics 2009, 9, 2108‐2117. Sharing your niche with an unrelated species Anyone who's ever lived with a roommate knows the pain of dividing up the refrigerator space and the cleaning duties as well as the rent. Is it based on number of people, the size of bedrooms, or size of biceps? Many “free‐living” bacteria share their living space with other species in stable consortia to which each member contributes. Bobadilla Fazzini et al. use proteomic and other tools to examine the changes resulting from shifts in limiting carbon sources. Their system is a continuous culture of 9:1 Pseudomonas reinekei (MT1): Achromobacter xylosoxidans (MT3), cultured from a contaminated stream and able to grow on 4‐chlorosalicylate, an intermediate in the degradation of toxic furans and dioxins. MT1 OprF, the outer membrane protein and homolog of E. coli OmpA, is a “slow porin” that contributes to toxin resistance. After a shift in carbon sources, MT1 OprF was up‐regulated ～11‐fold in mixed culture vs. pure culture. Bobadilla Fazzini, R. A. et al., Proteomics 2009, 9, 2273‐2285. Heart to heart: Biomarkers for MACE Mace is a spice, not an herb. It is a badge of office and a weapon (albeit now of a defensive sort). It is also an acronym for a Major Adverse Cardiac Event, otherwise known as a big heart attack, something you want to know is coming and to prevent. So what to do? Biomarkers to the rescue. Currently the FDA has approved one prospective test: the CardioMPO? ELISA test for myeloperoxidase. The MPO marker is >60% accurate in predicting a MACE over 30 days and 6 months. Zhou et al. propose an alternative statistical method for evaluating a panel of mass spectrometry markers. An improved preprocessing procedure utilizes low‐level signal processing and spectrum cleanup routines followed by partial least squares logistic regression and support vector machine classifier to select the markers. The prediction is done by an improved genetic algorithm with local optimization. Using seven markers yields >75% accuracy. Zhou, X. et al., Proteomics 2009, 9, 2286‐2294.     

Cover Picture: Proteomics 8/2009

In this issue of Proteomics you will find the following highlighted articles: Are you sick or are you just getting old: Does it matter? We all joke about the hazards of aging: various systems that break down, some you didn't even know you had. But then there's the alternative of not aging. Hmmm. Now what if aging were a disease? If so, it is worse than any cold I've had. Zürbig et al. have found that the proteome of the aging kidney has many markers in common with chronic kidney disease. The degree of match among small peptide markers ranged from 4% to 22% for IgA nephropathy to diabetic nephropathy, respectively. From these data they developed an age estimating scale that revealed some individuals had kidneys apparently “older” than their bodies. If these findings hold up, they could offer new approaches to diagnosis and therapy of chronic kidney diseases. Zürbig, P. et al., Proteomics 2009, 9, 2108‐2117. Sharing your niche with an unrelated species Anyone who's ever lived with a roommate knows the pain of dividing up the refrigerator space and the cleaning duties as well as the rent. Is it based on number of people, the size of bedrooms, or size of biceps? Many “free‐living” bacteria share their living space with other species in stable consortia to which each member contributes. Bobadilla Fazzini et al. use proteomic and other tools to examine the changes resulting from shifts in limiting carbon sources. Their system is a continuous culture of 9:1 Pseudomonas reinekei (MT1): Achromobacter xylosoxidans (MT3), cultured from a contaminated stream and able to grow on 4‐chlorosalicylate, an intermediate in the degradation of toxic furans and dioxins. MT1 OprF, the outer membrane protein and homolog of E. coli OmpA, is a “slow porin” that contributes to toxin resistance. After a shift in carbon sources, MT1 OprF was up‐regulated ～11‐fold in mixed culture vs. pure culture. Bobadilla Fazzini, R. A. et al., Proteomics 2009, 9, 2273‐2285. Heart to heart: Biomarkers for MACE Mace is a spice, not an herb. It is a badge of office and a weapon (albeit now of a defensive sort). It is also an acronym for a Major Adverse Cardiac Event, otherwise known as a big heart attack, something you want to know is coming and to prevent. So what to do? Biomarkers to the rescue. Currently the FDA has approved one prospective test: the CardioMPO? ELISA test for myeloperoxidase. The MPO marker is >60% accurate in predicting a MACE over 30 days and 6 months. Zhou et al. propose an alternative statistical method for evaluating a panel of mass spectrometry markers. An improved preprocessing procedure utilizes low‐level signal processing and spectrum cleanup routines followed by partial least squares logistic regression and support vector machine classifier to select the markers. The prediction is done by an improved genetic algorithm with local optimization. Using seven markers yields >75% accuracy. Zhou, X. et al., Proteomics 2009, 9, 2286‐2294.     

Crystal Structure and Catalytic Mechanism of 4-Methylmuconolactone Methylisomerase

When methyl-substituted aromatic compounds are degraded via ortho (intradiol)-cleavage of 4-methylcatechol, the dead-end metabolite 4-methylmuconolactone (4-ML) is formed. Degradation of 4-ML has only been described in few bacterial species, including Pseudomonas reinekei MT1. The isomerization of 4-ML to 3-methylmuconolactone (3-ML) is the first step required for the mineralization of 4-ML and is catalyzed by an enzyme termed 4-methylmuconolactone methylisomerase (MLMI). We identified the gene encoding MLMI in P. reinekei MT1 and solved the crystal structures of MLMI in complex with 3-ML at 1.4-Å resolution, with 4-ML at 1.9-Å resolution and with a MES buffer molecule at 1.45-Å resolution. MLMI exhibits a ferredoxin-like fold and assembles as a tight functional homodimeric complex. We were able to assign the active site clefts of MLMI from P. reinekei MT1 and of the homologous MLMI from Cupriavidus necator JMP134, which has previously been crystallized in a structural genomics project. Kinetic and structural analysis of wild-type MLMI and variants created by site-directed mutagenesis indicate Tyr-39 and His-26 to be the most probable catalytic residues. The previously proposed involvement of Cys-67 in covalent catalysis can now be excluded. Residue His-52 was found to be important for substrate affinity, with only marginal effect on catalytic activity. Based on these results, a novel catalytic mechanism for the isomerization of 4-ML to 3-ML by MLMI, involving a bislactonic intermediate, is proposed. This broadens the knowledge about the diverse group of proteins exhibiting a ferredoxin-like fold.     

ALLOPOLYPLOIDY IN NATURAL AND CULTIVATED POPULATIONS OF PORPHYRA (BANGIALES,RHODOPHYTA)1

To confirm whether allopolyploidy occurs in samples of previously identified Porphyra yezoensis Ueda, P. tenera Kjellm., and P. yezoensis × P. tenera from natural and cultivated populations, we examined these samples by using PCR‐RFLP and microsatellite analyses of multiple nuclear and chloroplast regions [nuclear regions: type II DNA topoisomerase gene (TOP2), actin‐related protein 4 gene (ARP4), internal transcribed spacer (ITS) rDNA and three microsatellite loci; chloroplast region: RUBISCO spacer]. Except for the ITS region, these multiple nuclear markers indicated that the wild strain MT‐1 and the cultivated strain 90‐02 (previously identified as P. yezoensis × P. tenera and cultivated P. tenera, respectively) are heterozygous and possess both genotypes of P. tenera and P. yezoensis in the conchocelis phase. Furthermore, gametophytic blades of two pure lines, HG‐TY1 and HG‐TY2 (F₁ strains of MT‐1 and 90‐02, respectively), were also heterozygous, and six chromosomes per single cell could be observed in each blade of the two pure lines. These results demonstrate that allopolyploidy occurs in Porphyra strains derived from both natural and cultivated populations, even though ITS genotypes of these strains showed homogenization toward one parental ITS.     

Tyrosol degradation via the homogentisic acid pathway in a newly isolated Halomonas strain from olive processing effluents

Aims: To isolate a new Halomonas sp. strain capable of degrading tyrosol, a toxic compound present in olive mill wastewater, through the homogentisic acid (HGA) pathway. Methods and Results: A moderately halophilic Gram‐negative bacterium belonging to the Halomonas genus and designated strain TYRC17 was isolated from olive processing effluents. This strain was able to completely degrade tyrosol (2‐(p‐hydroxyphenyl)‐ethanol), a toxic compound found in such effluent. Tyrosol degradation begins by an oxidation to 4‐hydroxyphenylacetic acid (HPA), which is then converted into HGA by an HPA 1‐monooxygenase, while closest Halomonas species degrade tyrosol through 3,4‐dihydroxyphenylacetic acid (DHPA). In the presence of transition metals, HGA underwent a pH‐dependent abiotic conversion into benzoquinone acetic acid, then into 2,5‐dihydroxybenzaldehyde (gentisaldehyde) and pyomelanin, by oxidative decarboxylation and polymerization, respectively. Conclusions: Tyrosol degradation via HGA by the new Halomonas sp. strain TYRC17 was complete in the absence of trace elements. In their presence, HGA was abiotically converted into gentisaldehyde and pyomelanin. Significance and Impact of the Study: This is the first report on tyrosol degradation via the HGA pathway under hypersaline conditions and on the oxidative decarboxylation of HGA into gentisaldehyde. It underlines the importance of the Halomonas genus in the bioremediation of toxic‐contaminated sites.     

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system‐dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N‐myristoylation motif is essential for its localization. Chemical‐induced expression of AvrXccB suppresses flg22‐triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S‐adenosyl‐l ‐methionine‐dependent methyltransferases SAM‐MT1 and SAM‐MT2. Interestingly, SAM‐MT1 is not only self‐associated, but also associated with SAM‐MT2 in vivo. SAM‐MT1 and SAM‐MT2 expression is significantly induced upon stimulation of microbe‐associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.     

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60‐1, was bred from a parental strain, MT8‐1, via stepwise adaptation. YK60‐1 grew at 40°C, a temperature at which MT8‐1 could not grow at all. YK60‐1 exhibited faster growth than MT8‐1 at 30°C. To investigate the mechanisms how MT8‐1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress‐responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60‐1. Furthermore, nontargeting metabolome analysis showed that YK60‐1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8‐1. In conclusion, S. cerevisiae MT8‐1 acquired thermotolerance by induction of specific stress‐responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1116–1123, 2013     

Biodegradation of endosulfan and endosulfan sulfate by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO₂ from the culture media with endosulfan as sulfur source as compared to MgSO₄ suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.     

Survival of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> Strains from Different Origins Under Oxidative Stress Conditions: Effect of Temperature

Campylobacter jejuni is a microaerophilic pathogen but is able to survive oxidative stress conditions during its transmission to the human host. Strains of different origins (reference, poultry, or human clinical) were tested for survival under oxidative stress conditions. C. jejuni strains were grown in Mueller Hinton broth to obtain late exponential–phase cultures. Then they were exposed to 2 different stresses: (1) cultures were either plated on Columbia agar plates and exposed to atmospheric oxygen or (2) paraquat (a chemical oxidizing agent) was added to liquid cultures to reach a 500-μM concentration. Both of these experimental conditions were realized at 3 different temperatures: 4°C, 25°C, and 42°C. Results obtained with paraquat and atmospheric oxygen were similar. Surprisingly, C. jejuni was found to be very sensitive to oxidative stress at 42°C, which is its optimal growth temperature, whereas it was more resistant at 4°C. A strain effect was observed, but no relationship was found between the origin of the strains and level of resistance. High temperature (42°C) combined with oxidative stress allowed a rapid decrease in the C. jejuni population, whereas low temperature considerably decreased the effect of oxidative stress.     

Ganoderma atrum polysaccharide protects cardiomyocytes against anoxia/reoxygenation‐induced oxidative stress by mitochondrial pathway

It is now well established that oxidative stress plays a causative role in the pathogenesis of anoxia/reoxygenation (A/R) injury. Ganoderma atrum polysaccharide (PSG‐1), the most abundant component isolated from G. atrum, has been shown to possess potent antioxidant activity. The goals of this study were to investigate the effect of PSG‐1 against oxidative stress induced by A/R injury and the possible mechanisms in cardiomyocytes. In this work, primary cultures of neonatal rat cardiomyocytes pretreated with PSG‐1 were subjected to A/R and subsequently monitored for cell viability by the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay. The levels of intracellular reactive oxygen species (ROS), apoptosis, and mitochondrial membrane potential (Δψ_m) were determined by flow cytometry. Western blot analysis was used to measure the expression of cytochrome c, Bcl‐2 family, and manganese superoxide dismutase (MnSOD) proteins, and the activities of caspase‐3 and caspase‐9 were determined by a colorimetric method. The results showed that PSG‐1 protected against cell death caused by A/R injury in cardiomyocytes. PSG‐1 reduced the A/R‐induced ROS generation, the loss of mitochondrial membrane potential (Δψ_m), and the release of cytochrome c from the mitochondria into cytosol. PSG‐1 inhibited the A/R‐stimulated activation of caspase‐9 and caspase‐3 and alteration of Bcl‐2 family proteins. Moreover, PSG‐1 significantly increased the protein expression of MnSOD in cardiomyocytes. These findings suggest that PSG‐1 significantly attenuates A/R‐induced oxidative stress and improves cell survival in cardiomyocytes through mitochondrial pathway. J. Cell. Biochem. 110: 191–200, 2010. © 2010 Wiley‐Liss, Inc.     

The cry1Ac gene of Bacillus thuringiensis ZQ-89 encodes a toxin against long-horned beetle adult

Aims: Bacillus thuringiensis is toxic to many insects including Coleopteran pests. However, there is no report that B. thuringiensis is toxic to the adults of long‐horned beetle, Batocera horsfieldi, a pest of poplar trees. This work aims to select a B. thuringiensis strain toxic towards the adults of Asian long‐horned beetle. Methods and Results: A total of 504 B. thuringiensis strains were tested for the insecticidal activity to B. horsfieldi adults by artificial feeding. A strain of ZQ‐89 was found with a high toxicity to B. horsfieldi adults. The rectified lethal rate of ZQ‐89 to beetle was 55·33%. Additionally, the body weight and egg‐hatching rate of beetle, respectively, decreased by 2·22 and 19·62% after being fed with ZQ‐89. Further investigation found that the pure parasporal crystal had high toxicity to beetle adults. The ZQ‐89 crystal protein was purified and analysed by peptide‐mapping fingerprint and found it was highly homologous to Cry1Ac protein. The crystal protein gene was cloned and named cry1Ac89. The cry1Ac89 gene and its promoter were inserted into the plasmid pHT304 and then transformed into B. thuringiensis acrystalliferous strain BMB171. SDS‐PAGE analysis showed the BMB171‐Cry1Ac89 recombinant strain successfully expresses a 133‐kDa recombinant crystal protein with highly lethal activity to B. horsfieldi adults. Conclusions: The strain of ZQ‐89 is highly toxic to the adults of long‐horned beetle, and the crystal protein mainly contributes to the antipest role of this strain. The cry1Ac89 gene is a good candidate to be used for making transgenic trees or develop environment‐friendly bioinsecticides against long‐horned beetles such as B. horsfieldi in the future. Significance and Impact of the Study: It is the first report of a B. thuringiensis strain toxic to the adults of Asian long‐horned beetle, and the Cry1Ac protein is also firstly reported to be toxic to Coleopteran pests.     

Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts

The relationship between drought, oxidative stress and leaf senescence was evaluated in field‐grown sage (Salvia officinalis L.), a drought‐susceptible species that shows symptoms of senescence when exposed to stress. Despite the photoprotection conferred by the xanthophyll cycle, drought‐stressed senescing leaves showed enhanced lipid peroxidation, chlorophyll loss, reduced photosynthetic activity and strong reductions of membrane‐bound chloroplastic antioxidant defences (i.e. β‐carotene and α‐tocopherol), which is indicative of oxidative stress in chloroplasts. H₂O₂ accumulated in drought‐stressed senescing leaves. Subcellular localization studies showed that H₂O₂ accumulated first in xylem vessels and the cell wall and later in the plasma membrane of mesophyll cells, but not in chloroplasts, indicating reactive oxygen species other than H₂O₂ as direct responsible for the oxidative stress observed in the chloroplasts of drought‐stressed senescing leaves. The strong degradation of β‐carotene and α‐tocopherol suggests an enhanced formation of singlet oxygen as the putative reactive oxygen species responsible for oxidative stress to senescing chloroplasts. This study demonstrates that oxidative stress in chloroplasts mediates drought‐induced leaf senescence in sage growing in Mediterranean field conditions.     

Characterization of Fluorescent Siderophore-Mediated Iron Uptake in Pseudomonas sp. Strain M114: Evidence for the Existence of an Additional Ferric Siderophore Receptor

In Pseudomonas sp. strain M114, the outer membrane receptor for ferric pseudobactin M114 was shown to transport ferric pseudobactins B10 and A225, in addition to its own. The gene encoding this receptor, which was previously cloned on pCUP3, was localized by Tn5 mutagenesis to a region comprising >1.6 kb of M114 DNA. A mutant (strain M114R1) lacking this receptor was then created by a marker exchange technique. Characterization of this mutant by using purified pseudobactin M114 in radiolabeled ferric iron uptake studies confirmed that it was completely unable to utilize this siderophore for acquisition of iron. In addition, it lacked an outer membrane protein band of 89 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, growth of the mutant was severely restricted under low-iron conditions. However, this phenotype was reversed in the presence of another fluorescent siderophore (pseudobactin MT3A) from Pseudomonas sp. strain MT3A, suggesting the presence of a second receptor in strain M114. Furthermore, wild-type Pseudomonas sp. strain B24 was not able to utilize ferric pseudobactin MT3A, and this phenotype was not reversed upon expression of the M114 receptor encoded on pCUP3. However, a cosmid clone (pMS1047) that enabled strain B24 to utilize ferric pseudobactin MT3A was isolated from an M114 gene bank. Radiolabel transport assays with purified pseudobactin MT3A confirmed this event. Plasmid pMS1047 was shown to encode an outer membrane protein of 81 kDa in strain B24 under iron-limiting conditions; this protein corresponds to a similar protein in strain M114.     

Menaquinone biosynthesis potentiates haem toxicity in Staphylococcus aureus

Staphylococcus aureus is a pathogen that infects multiple anatomical sites leading to a diverse array of diseases. Although vertebrates can restrict the growth of invading pathogens by sequestering iron within haem, S. aureus surmounts this challenge by employing high‐affinity haem uptake systems. However, the presence of excess haem is highly toxic, necessitating tight regulation of haem levels. To overcome haem stress, S. aureus expresses the detoxification system HrtAB. In this work, a transposon screen was performed in the background of a haem‐susceptible, HrtAB‐deficient S. aureus strain to identify the substrate transported by this putative pump and the source of haem toxicity. While a recent report indicates that HrtAB exports haem itself, the haem‐resistant mutants uncovered by the transposon selection enabled us to elucidate the cellular factors contributing to haem toxicity. All mutants identified in this screen inactivated the menaquinone (MK) biosynthesis pathway. Deletion of the final steps of this pathway revealed that quinone molecules localizing to the cell membrane potentiate haem‐associated superoxide production and subsequent oxidative damage. These data suggest a model in which membrane‐associated haem and quinone molecules form a redox cycle that continuously generates semiquinones and reduced haem, both of which react with atmospheric oxygen to produce superoxide.     

SiaA and SiaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa

Cell aggregation is a stress response and serves as a survival strategy for Pseudomonas aeruginosa strain PAO1 during growth with the toxic detergent Na‐dodecylsulfate (SDS). This process involves the psl operon and is linked to c‐di‐GMP signalling. The induction of cell aggregation in response to SDS was studied. Transposon and site‐directed mutagenesis revealed that the cupA‐operon and the co‐transcribed genes siaA (PA0172) and siaD (PA0169) were essential for SDS‐induced aggregation. While siaA encodes a putative membrane protein with a HAMP and a PP2C‐like phosphatase domain, siaD encodes a putative diguanylate cyclase involved in the biosynthesis of c‐di‐GMP. Complementation studies uncovered that the loss of SDS‐induced aggregation in the formerly isolated spontaneous mutant strain N was caused by a non‐functional siaA allele. DNA‐microarray analysis of SDS‐grown cells revealed consistent activation of eight genes, including cupA1, with known or presumptive important functions in cell aggregation in the parent strain compared with non‐aggregating siaA and siaD mutants. A siaAD‐dependent increase of cupA1 mRNA levels in SDS‐grown cells was also shown by Northern blots. These results clearly demonstrate that SiaAD are essential for inducing cell aggregation as a specific response to SDS and suggest that they are responsible for perceiving and transducing SDS‐related stress.