Jens Glaeser

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 (1O2) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to 1O2. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with 1O2 or superoxide (O–2). One sRNA was specifically induced by 1O2 and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by 1O2 and O–2 were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoHI and RpoHII. The most abundant sRNA was processed in the presence of 1O2 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 1O2‐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 1O2 exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.

Singlet Oxygen Stress in Microorganisms

Singlet oxygen is the primary agent of photooxidative stress in microorganisms. In photosynthetic microorganisms, sensitized generation by pigments of the photosystems is the main source of singlet oxygen and, in nonphotosynthetic microorganisms, cellular cofactors such as flavins, rhodopsins, quinones, and porphyrins serve as photosensitizer. Singlet oxygen rapidly reacts with a wide range of cellular macromolecules including proteins, lipids, DNA, and RNA, and thereby further reactive substances including organic peroxides and sulfoxides are formed. Microorganisms that face high light intensities or exhibit potent photosensitizers have evolved specific mechanisms to prevent photooxidative stress. These mechanisms include the use of quenchers, such as carotenoids, which interact either with excited photosensitizer molecules or singlet oxygen itself to prevent damage of cellular molecules. Scavengers like glutathione react with singlet oxygen. Despite those protection mechanisms, damage by reactions with singlet oxygen on cellular macromolecules disturbs cellular functions. Microorganisms that regularly face photooxidative stress have evolved specific systems to sense singlet oxygen and tightly control the removal of singlet oxygen reaction products. Responses to photooxidative stress have been investigated in a range of photosynthetic and nonphotosynthetic microorganisms. However, detailed knowledge on the regulation of this response has only been obtained for the phototrophic alpha-proteobacterium Rhodobacter sphaeroides. In this organism and in related proteobacteria, the extracytoplasmic function (ECF) sigma factor RpoE is released from the cognate antisigma factor ChrR in the presence of singlet oxygen and triggers the expression of genes providing protection against photooxidative stress. Recent experiments show that singlet oxygen acts as a signal, which is sensed by yet unknown components and leads to proteolysis of ChrR. RpoE induces expression of a second alternative sigma factor, RpoH(II), which controls a large set of genes that partially overlaps with the heat-shock response controlled by RpoH(I). In addition to the transcriptional control of gene regulation by alternative sigma factors, a set of noncoding small RNAs (sRNAs) appear to affect the synthesis of several proteins involved in the response to photooxidative stress. The interaction of mRNA targets with those sRNAs is usually mediated by the RNA chaperone Hfq. Deletion of the gene encoding Hfq leads to a singlet oxygen-sensitive phenotype, which underlines the control of gene regulation on the posttranscriptional level by sRNAs in R. sphaeroides. Hence, a complex network of different regulatory components controls the defense against photooxidative stress in anoxygenic photosynthetic bacteria.

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 organisms defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoH(II) in R. sphaeroides.

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 (1O2). Exposure to 1O2 induces the alternative sigma factors RpoE and RpoHII 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 1O2. The higher 1O2 sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoHII‐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 1O2‐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.

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.

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.

Novosphingobium acidiphilum sp. nov., an acidophilic salt-sensitive bacterium isolated from the humic acid-rich Lake Grosse Fuchskuhle

A yellow- to orange-pigmented, Gram-negative, rod-shaped, motile and non-spore-forming bacterium, strain FSW06-204d(T), was isolated from subsurface water of the acidic bog lake, Lake Grosse Fuchskuhle (Brandenburg, Germany). Optimum growth of this strain occurred over a pH range from 5.5 to 6.0 and the growth rate strongly decreased at pH values above 6.5. In addition, the strain exhibited a low tolerance towards NaCl and grew only at a NaCl concentration of up to 0.5 %. 16S rRNA gene sequence analysis of strain FSW06-204d(T) showed the highest sequence similarity to Novosphingobium hassiacum W-51(T) (96.7 %) and formed a distinct cluster with Novosphingobium nitrogenifigens DSM 19370(T) (96.4 %) within the genus Novosphingobium. Strain FSW06-204d(T) shared a 21 bp signature gap with the latter species, a feature that is absent in all other members of the family Sphingomonadaceae. DNA-DNA hybridization of strain FSW06-204d(T) and N. nitrogenifigens DSM 19370(T) showed a low relatedness value of 24 % (reciprocal: 39 %). The major respiratory quinone was ubiquinone Q-10 (91 %) and the predominant fatty acid was C(18 : 1)omega7c (43.3 %). Two characteristic 2-hydroxy fatty acids, C(14 : 0) 2-OH (8.1 %) and C(15 : 0) 2-OH (6.5 %), were abundant. Polar lipids consisted mainly of phosphatidyldimethylethanolamine and phosphatidylethanolamine; however, only moderate amounts of sphingoglycolipids were present and phosphatidylcholine was lacking. Characterization by 16S rRNA gene sequence, physiological features, pigment analysis and polyamine, ubiquinone, polar lipid and fatty acid contents revealed that strain FSW06-204d(T) represents a novel species of the genus Novosphingobium within the class Alphaproteobacteria. The name Novosphingobium acidiphilum sp. nov. is proposed for this acidophilic and salt-sensitive species with the type strain FSW06-204d(T) (=DSM 19966(T)=CCM 7496(T)=CCUG 55538(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.

DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides

Singlet oxygen (1O2) 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 1O2 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 1O2 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 1O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to 1O2 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 1O2 exposure in R. sphaeroides was extended.

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 (1O2) and hydrogen peroxide (H2O2). Because these ROS differ in half-life time and toxicity, we compared their effects on microbial activity (14C-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 1O2 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 1O2 exposure, but decreased after H2O2 exposure. The opposite pattern was found for Actinobacteria of the freshwater AcI-B cluster which were highly sensitive to 1O2 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 1O2 exposure compared to free-living populations. These results imply that 1O2 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.