Trichoderma-Derived Pentapeptides from the Infected Nest Mycobiome of the Subterranean Termite Coptotermes testaceus.

Termites live in a dynamic environment where colony health is strongly influenced by surrounding microbes. However, little is known about the mycobiomes of lower termites and their nests, and how these change in response to disease. Here we compared the individual and nest mycobiomes of a healthy subterranean termite colony (Coptotermes testaceus) to one infected and ultimately eradicated by a fungal pathogen. We identified Trichoderma species in the materials of both nests, but they were also abundant in the infected termites. Methanolic extracts of Trichoderma sp. FHG000531, isolated from the infected nest, were screened for secondary metabolites by UHPLC-HR MS/MS-guided molecular networking. We identified many bioactive compounds with potential roles in the eradication of the infected colony, as well as a cluster of six unknown peptides. The novel peptide FE011 was isolated and characterized by NMR spectroscopy. The function of this novel peptide family as well as the role of Trichoderma species in dying termite colonies therefore requires further investigation.

Molecular Networking-Guided Discovery and Characterization of Stechlisins, a Group of Cyclic Lipopeptides from a Pseudomonas sp.

Increasingly sensitive analytical instruments and robust downstream data processing tools have revolutionized natural product research over the past decade. A molecular networking-guided survey led to the identification of 33 new cyclic lipopeptides (CLPs) from the culture broth of the proteobacterium Pseudomonas sp. FhG100052. The compound family resembles members of the amphisin group of CLPs that possess a 3-hydroxy fatty acid linked to the N-terminus of an undecapeptide core. Culture optimization led to the isolation and subsequent structure elucidation of one known and five new derivatives by extensive MS/MS and NMR experiments in combination with Marfey's analysis. The data were in agreement with in silico analysis of the corresponding biosynthetic gene cluster. Most strikingly, the length of the incorporated fatty acid defined the growth inhibitory effects against Moraxella catarrhalis FH6810, as observed by MIC values ranging from no inhibition (>128 µg/mL) to 4 µg/mL.

Regulation of a polyamine transporter by the conserved 3' UTR-derived sRNA SorX confers resistance to singlet oxygen and organic hydroperoxides in Rhodobacter sphaeroides.

Singlet oxygen is generated by bacteriochlorophylls when light and oxygen are simultaneously present in Rhodobacter sphaeroides. Singlet oxygen triggers a specific response that is partly regulated by the alternative sigma factor RpoHI/HII. The sRNA RSs2461 has previously been identified as an RpoHI/HII-dependent sRNA and is derived from the 3' UTR of the mRNA for an OmpR-type transcriptional regulator. Similar to the RpoHI/HII-dependent CcsR and SorY sRNAs, RSs2461 affects the resistance of R. sphaeroides against singlet oxygen and was therefore renamed here SorX. Furthermore, SorX has a strong impact on resistance against organic hydroperoxides that usually occur as secondary damages downstream of singlet oxygen. The 75-nt SorX 3' fragment, which is generated by RNase E cleavage and highly conserved among related species, represents the functional entity. A target search identified potA mRNA, which encodes a subunit of a polyamine transporter, as a direct SorX target and stress resistance via SorX could be linked to potA. The PotABCD transporter is an uptake system for spermidine in E. coli. While spermidine is generally described as beneficial during oxidative stress, we observed significantly increased sensitivity of R. sphaeroides to organic hydroperoxides in the presence of spermidine. We therefore propose that the diminished import of spermidine, due to down-regulation of potA by SorX, counteracts oxidative stress. Together with results from other studies this underlines the importance of regulated transport to bacterial stress defense.

Novosphingobium aquaticum sp. nov., isolated from the humic-matter-rich bog lake Grosse Fuchskuhle.

A yellow-pigmented, Gram-negative rod, designated FNE08-86(T), was isolated from subsurface water of the humic-matter-rich and almost-neutral north-east basin of the experimentally divided bog lake Grosse Fuchskuhle (Brandenburg, Germany). Analysis of the nearly full-length 16S rRNA gene sequence showed the highest 16S rRNA gene sequence similarity with Novosphingobium rosa IAM 14222(T) (96.3 %). Sequence similarities with all other members of the genus Novosphingobium species were <96 %, but phylogenetic tree construction clearly showed the placement of strain FNE08-86(T) within the genus Novosphingobium. The predominant fatty acids were C18 : 1ω7c and C16 : 0, and only a single 2-hydroxy fatty acid, C14 : 0 2-OH, was detected. The polar lipid profile revealed phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine as major compounds, with smaller amounts of sphingoglycolipid, phosphatidylmonomethylethanolamine, diphosphatidylglycerol and several unidentified lipids. In the quinone system ubiquinone Q-10 was predominant and in the polyamine pattern spermidine was predominant. Characterization by genotypic, chemotaxonomic and phenotypic analysis indicated that strain FNE08-86(T) represents a novel species of the genus Novosphingobium, for which we propose the name Novosphingobium aquaticum sp. nov. (type strain FNE08-86(T) = DSM 25088(T) = CCM 7983(T)).

Novosphingobium fuchskuhlense sp. nov., isolated from the north-east basin of Lake Grosse Fuchskuhle.

A yellow pigmented, gram-negative, rod-shaped bacterium designated FNE08-7(T) was isolated from subsurface water of the north-east basin of the bog lake Grosse Fuchskuhle (Brandenburg, Germany). A first analysis of the nearly full-length 16S rRNA gene sequence analysis including environmental 16S rRNA gene sequences derived from freshwater ecosystems showed that strain FNE08-7(T) is the first cultured representative, to our knowledge, of the freshwater tribe Novo-A2. Further analysis indicates highest 16S rRNA gene sequence similarities to the type strains of Novosphingobium stygium (98.0 %) and Novosphingobium taihuense (97.4 %) and between 94.0 % and 96.9 % sequence similarity to other members of the genus Novosphingobium. Reconstruction of phylogenetic trees showed that strain FNE08-7(T) formed a distinct cluster with the type strains of N. stygium and N. taihuense supported by high bootstrap values. DNA-DNA hybridization of strain FNE08-7(T) with N. stygium SMCC B0712(T) and N. taihuense DSM 17507(T) revealed low similarity values of 18.4 % (reciprocal: 11.4 %) and 23.1 % (reciprocal: 54.2 %), respectively. The predominant fatty acid of the isolate is C(18 : 1)ω7c (56.4 %) and two characteristic 2-hydroxy fatty acids, C(14 : 0) 2-OH (16.5 %) and C(15 : 0) 2-OH (3.3 %) occur. Ubiquinone Q-10 is the major respiratory quinone. The predominant polar lipids are phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, sphingoglycolipid, phosphatidylcholine and minor amounts of diphosphatidylglycerol. Spermidine is the predominant polyamine. Characterization by genotypic, chemotaxonomic and phenotypic analysis indicate that strain FNE08-7(T) represents a novel species of the genus Novosphingobium within the Alphaproteobacteria. Therefore, we propose the species Novosphingobium fuchskuhlense sp. nov., with FNE08-7(T) ( = DSM 25065(T) = CCM 7978(T) = CCUG 61508(T)) as the type strain.

Contribution of Hfq to photooxidative stress resistance and global regulation in Rhodobacter sphaeroides.

The photosynthetic alphaproteobacterium Rhodobacter sphaeroides has to cope with photooxidative stress that is caused by the bacteriochlorophyll a-mediated formation of singlet oxygen ((1)O(2)). Exposure to (1)O(2) induces the alternative sigma factors RpoE and RpoH(II) which then promote transcription of photooxidative stress-related genes, including small RNAs (sRNAs). The ubiquitous RNA chaperone Hfq is well established to interact with and facilitate the base-pairing of sRNAs and target mRNAs to influence mRNA stability and/or translation. Here we report on the pleiotropic phenotype of a Δhfq mutant of R. sphaeroides, which is less pigmented, produces minicells and is more sensitive to (1)O(2). The higher (1)O(2) sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoH(II)-dependent genes. We used co-immunoprecipitation of FLAG-tagged Hfq combined with RNA-seq to identify association of at least 25 sRNAs and of mRNAs encoding cell division proteins and ribosomal proteins with Hfq. Remarkably, > 70% of the Hfq-bound sRNAs are (1)O(2)-affected. Proteomics analysis of the Hfq-deficient strain revealed an impact of Hfq on amino acid transport and metabolic functions. Our data demonstrate for the first time an involvement of Hfq in regulation of photosynthesis genes and in the photooxidative stress response.

Genomic and Chemical Decryption of the Bacteroidetes Phylum for Its Potential to Biosynthesize Natural Products.

With progress in genome sequencing and data sharing, 1,000s of bacterial genomes are publicly available. Genome mining-using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis, and rating-has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Mining this data set, we isolated the new iron chelating nonribosomally synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively. IMPORTANCE The development of pipelines for anti-infectives to be applied in plant, animal, and human health management are dried up. However, the resistance development against compounds in use calls for new lead structures. To fill this gap and to enhance the probability of success for the discovery of new bioactive natural products, microbial taxa currently underinvestigated must be mined. This study investigates the potential within the bacterial phylum Bacteroidetes. A combination of omics-technologies revealed taxonomical hot spots for specialized metabolites. Genome- and metabolome-based analyses showed that the phylum covers a new chemical space compared with classic natural product producers. Members of the Bacteroidetes may thus present a promising bioresource for future screening and isolation campaigns.

Combination of high-throughput microfluidics and FACS technologies to leverage the numbers game in natural product discovery.

High-throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence-activated cell sorting (FACS)-based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC-QTOF-MS/MS-guided fractionation and microplate-based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.

Anoxygenic photosynthesis and photooxidative stress: a particular challenge for Roseobacter.

Roseobacter clade aerobic anoxygenic phototrophic bacteria (AAnP) are abundant in photic zone environments of marine ecosystems. These bacteria form a photosynthetic apparatus at oxygen saturation, a situation expected to generate high levels of singlet oxygen (¹O2) when light is present. Rhodobacter sphaeroides, an anaerobic anoxygenic phototroph, represses photosynthesis genes at high oxygen tension. Here we report that Roseobacter denitrificans showed higher sensitivity to ¹O2 compared with Rhb. sphaeroides. While photosynthetic membranes of Rsb. denitrificans generated more ¹O2 during light exposure, key regulator genes rpoE and rpoH(II) were more strongly induced in response to ¹O2 stress compared with Rhb. sphaeroides. The regulon controlled by RpoE was different in Rsb. denitrificans and Rhb. sphaeroides. Patterns of synthesized soluble proteins strongly changed upon high light exposure in Rsb. denitrificans but not in Rhb. sphaeroides, and most changes were not further promoted by artificial ¹O2 generation. The strong increase of small RNA RDs2461 levels by photooxidative stress implies a role for sRNAs in post-transcriptional regulation of the response to ¹O2 in AAnPs. Our data reveal similarities but also significant differences in the response of Rsb. denitrificans and Rhb. sphaeroides to ¹O2, most likely a consequence of their different lifestyles.

The RSP_2889 gene product of Rhodobacter sphaeroides is a CueR homologue controlling copper-responsive genes.

Metal homeostasis is important in all living cells in order to provide sufficient amounts of metal ions for biological processes but to prevent toxic effects by excess amounts. Here we show that the gene product of RSP_2889 of the facultatively photosynthetic bacterium Rhodobacter sphaeroides is homologous to CueR, a regulator of copper metabolism in Escherichia coli and other bacteria. CueR binds to the promoter regions of genes for a copper-translocating ATPase and for a copper chaperone and is responsible for their high expression when cells are exposed to elevated levels of copper ions. While deletion of RSP_2889 has no significant effect on copper resistance, expression from a low-copy-number plasmid mediates increased sensitivity to copper.

Two-step generation of monodisperse agarose-solidified double emulsions (w/w/o) excluding an inner oil barrier.

Miniaturization of biomedical and chemical research areas is performed using microfluidic techniques. Droplet-based microfluidic applications are of high interest for various applications, e.g., high-throughput screening assays. Many of them are based on simple water-in-oil (w/o) or oil-in-water (o/w) emulsions that are easily to produce. More complex assays based on separate compartments require the use of multiple emulsions, such as water-in-oil-in-water (w/o/w) or oil-in-water-in-oil (o/w/o) emulsions. In this study an easy, fast to establish method to generate agarose-solidified (w/w/o) double emulsions with ∼55 µm in diameter, in which both agarose-phases are not separated by a surfactant stabilized oil is described. An off-chip emulsion-breaking and washing step of the inner agarose droplets based on density gradient centrifugation was designed, offering new possibilities for high-throughput assays on picoliter scale. In brief, this paper reports:•the protocol to generate agarose-solidified (w/w/o) double emulsions non-seperated by surfactant stabilized oil;•an off-chip washing protocol of agarose-solidified emulsions based on density gradient centrifugation.

Overlapping alternative sigma factor regulons in the response to singlet oxygen in Rhodobacter sphaeroides.

Organisms performing photosynthesis in the presence of oxygen have to cope with the formation of highly reactive singlet oxygen ((1)O(2)) and need to mount an adaptive response to photooxidative stress. Here we show that the alternative sigma factors RpoH(I) and RpoH(II) are both involved in the (1)O(2) response and in the heat stress response in Rhodobacter sphaeroides. We propose RpoH(II) to be the major player in the (1)O(2) response, whereas RpoH(I) is more important for the heat stress response. Mapping of the 5' ends of RpoH(II)- and also RpoH(I)/RpoH(II)-dependent transcripts revealed clear differences in the -10 regions of the putative promoter sequences. By using bioinformatic tools, we extended the RpoH(II) regulon, which includes genes induced by (1)O(2) exposure. These genes encode proteins which are, e.g., involved in methionine sulfoxide reduction and in maintaining the quinone pool. Furthermore, we identified small RNAs which depend on RpoH(I) and RpoH(II) and are likely to contribute to the defense against photooxidative stress and heat stress.

Photooxidative stress-induced and abundant small RNAs in Rhodobacter sphaeroides.

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen ((1)O(2)) in Rhodobacter sphaeroides. Our study reports the genome-wide search for small RNAs (sRNAs) involved in the regulatory response to (1)O(2). By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with (1)O(2) or superoxide (O(-)(2)). One sRNA was specifically induced by (1)O(2) and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by (1)O(2) and O(-)(2) were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH(I) and RpoH(II). The most abundant sRNA was processed in the presence of (1)O(2) but not by O(-)(2). From this and a second sRNA a conserved 3'-segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half-lives, abundance and processing of (1)O(2)-affected sRNAs. Orthologues of three sRNA genes are present in different alpha-proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by (1)O(2) exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.

Singlet oxygen, a neglected but important environmental factor: short-term and long-term effects on bacterioplankton composition in a humic lake.

Photolysis of dissolved organic matter (DOM) leads to contrasting effects on bacterioplankton dynamics, i.e. stimulation and inhibition of bacterial activity. In particular, the role of short-lived reactive oxygen species (ROS), e.g. singlet oxygen (¹O2), in altering microbial activity and species composition has scarcely been investigated. Therefore, we have artificially increased the natural rate of ¹O2 formation in short-term (∼4 h) in situ and long-term (72 h) laboratory incubations of surface water samples from a humic acid-rich lake. Denaturing gradient gel electrophoresis (DGGE) patterns revealed significant changes in occurrence of abundant bacterioplankton phylotypes upon ¹O2 exposure. Cluster analysis of DGGE patterns showed that a moderate increase in ¹O2 exposure leads to similar changes in different years indicating the establishment of bacterial communities adapted to ¹O2 exposure. Bacterioplankton phylotypes favoured under these conditions belonged to Betaproteobacteria of the beta II cluster (e.g. Polynucleobacter necessarius) and the beta I cluster related to Limnohabitans (R-BT subcluster) as well as Alphaproteobacteria affiliated to Novosphingobium acidiphilum. In contrast, Actinobacteria of the freshwater acI-B cluster were sensitive even against moderate ¹O2 exposure. We conclude that ¹O2 exposure due to DOM photolysis represents an important natural selective factor affecting bacterial species dynamics in aquatic ecosystems in many ways.

High-Throughput Cultivation for the Selective Isolation of Acidobacteria From Termite Nests.

Microbial communities in the immediate environment of socialized invertebrates can help to suppress pathogens, in part by synthesizing bioactive natural products. Here we characterized the core microbiomes of three termite species (genus Coptotermes) and their nest material to gain more insight into the diversity of termite-associated bacteria. Sampling a healthy termite colony over time implicated a consolidated and highly stable microbiome, pointing toward the fact that beneficial bacterial phyla play a major role in termite fitness. In contrast, there was a significant shift in the composition of the core microbiome in one nest during a fungal infection, affecting the abundance of well-characterized Streptomyces species (phylum Actinobacteria) as well as less-studied bacterial phyla such as Acidobacteria. High-throughput cultivation in microplates was implemented to isolate and identify these less-studied bacterial phylogenetic group. Amplicon sequencing confirmed that our method maintained the bacterial diversity of the environmental samples, enabling the isolation of novel Acidobacteriaceae and expanding the list of cultivated species to include two strains that may define new species within the genera Terracidiphilus and Acidobacterium.

RpoH(II) activates oxidative-stress defense systems and is controlled by RpoE in the singlet oxygen-dependent response in Rhodobacter sphaeroides.

Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen ((1)O(2)). Here we show that the alternative sigma factor RpoH(II) is required for the expression of important defense factors and that deletion of rpoH(II) leads to increased sensitivity against exposure to (1)O(2) and methylglyoxal in Rhodobacter sphaeroides. The gene encoding RpoH(II) is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an RpoH(II)-type sigma factor, which is widely distributed in the Alphaproteobacteria. RpoH(II)-dependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides, (1)O(2) scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organism's defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoH(II) in R. sphaeroides.

A response regulator of the OmpR family is part of the regulatory network controlling the oxidative stress response of Rhodobacter sphaeroides.

As a free-living bacterium Rhodobacter sphaeroides needs to respond to many environmental stresses. Oxidative stress, membrane stress or heat stress induce the ompR-1 gene encoding a protein of the OmpR family. Overexpression of OmpR-1 results in increased resistance to organic peroxides and diamide. Our data demonstrate that OmpR-1 positively affects expression of several sRNAs with an established role in R. sphaeroides stress defences and negatively affects the promoter of the rpoHI gene. The RpoHI sigma factor has a main role in the activation of many stress responses. Thus OmpR-1 has a balancing effect on the activation of the RpoHI regulon. We present a model with OmpR-1 as part of a regulatory network controlling stress defences in R. sphaeroides.

Subfossil 16S rRNA gene sequences of green sulfur bacteria in the Black Sea and their implications for past photic zone anoxia.

The Black Sea is the largest extant anoxic water body on Earth. Its oxic-anoxic boundary is located at a depth of 100 m and is populated by a single phylotype of marine green sulfur bacteria. This organism, Chlorobium sp. strain BS-1, is extraordinarily low light adapted and can therefore serve as an indicator of deep photic zone anoxia (A. K. Manske, J. Glaeser, M. M. M. Kuypers, and J. Overmann, Appl. Environ. Microbiol. 71:8049-8060, 2005). In the present study, two sediment cores were retrieved from the bottom of the Black Sea at depths of 2,006 and 2,162 m and were analyzed for the presence of subfossil DNA sequences of BS-1 using ancient-DNA methodology. Using optimized cultivation media, viable cells of the BS-1 phylotype were detected only at the sediment surface and not in deeper layers. In contrast, green sulfur bacterial 16S rRNA gene fragments were amplified from all the sediment layers investigated, including turbidites. After separation by denaturing gradient gel electrophoresis and sequencing, 14 different sequence types were distinguished. The sequence of BS-1 represented only a minor fraction of the amplification products and was found in 6 of 22 and 4 of 26 samples from the 2,006- and 2,162-m stations, respectively. Besides the sequences of BS-1, three additional phylotypes of the marine clade of green sulfur bacteria were detected. However, the majority of sequences clustered with groups from freshwater habitats. Our results suggest that a considerable fraction of green sulfur bacterial chemofossils did not originate in a low-light marine chemocline environment and therefore were likely to have an allochthonous origin. Thus, analysis of subfossil DNA sequences permits a more differentiated interpretation and reconstruction of past environmental conditions if specific chemofossils of stenoec species, like Chlorobium sp. strain BS-1, are employed.

Contrasting effects of singlet oxygen and hydrogen peroxide on bacterial community composition in a humic lake.

Light excitation of humic matter generates reactive oxygen species (ROS) in surface waters of aquatic ecosystems. Abundant ROS generated in humic matter rich lakes include singlet oxygen ((1)O2) and hydrogen peroxide (H2O2). Because these ROS differ in half-life time and toxicity, we compared their effects on microbial activity ((14)C-Leucine incorporation) and bacterial community composition (BCC) in surface waters of humic Lake Grosse Fuchskuhle (North-eastern Germany). For this purpose, experiments with water samples collected from the lake were conducted in July 2006, September 2008 and August 2009. Artificially increased (1)O2 and H2O2 concentrations inhibited microbial activity in water samples to a similar extent, but the effect of the respective ROS on BCC varied strongly. BCC analysis by 16S rRNA gene clone libraries and RT-PCR DGGE revealed ROS specific changes in relative abundance and activity of major bacterial groups and composition of dominating phylotypes. These changes were consistent in the three experiments performed in different years. The relative abundance of Polynucleobacter necessarius, Limnohabitans-related phylotypes (Betaproteobacteria), and Novosphingobium acidiphilum (Alphaproteobacteria) increased or was not affected by photo-sensitized (1)O2 exposure, but decreased after H2O2 exposure. The opposite pattern was found for Actinobacteria of the freshwater AcI-B cluster which were highly sensitive to (1)O2 but not to H2O2 exposure. Furthermore, group-specific RT-PCR DGGE analysis revealed that particle-attached P. necessarius and Limnohabitans-related phylotypes exhibit higher resistance to (1)O2 exposure compared to free-living populations. These results imply that (1)O2 acts as a factor in niche separation of closely affiliated Polynucleobacter and Limnohabitans-related phylotypes. Consequently, oxidative stress caused by photochemical ROS generation should be regarded as an environmental variable determining abundance, activity, and phylotype composition of environmentally relevant bacterial groups, in particular in illuminated and humic matter rich waters.

DegS and RseP homologous proteases are involved in singlet oxygen dependent activation of RpoE in Rhodobacter sphaeroides.

Singlet oxygen ((1)O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to (1)O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under (1)O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards (1)O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to (1)O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to (1)O2 exposure in R. sphaeroides was extended.