A tricistronic heat shock operon is important for stress tolerance of Pseudomonas putida and conserved in many environmental bacteria.

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Environmental Microbiology (2014) 16(6), 1835–1853

doi:10.1111/1462-2920.12432

A tricistronic heat shock operon is important for stress tolerance of Pseudomonas putida and conserved in many environmental bacteria

Stefanie S. Krajewski, Matthias Joswig, Miriam Nagel and Franz Narberhaus* Microbial Biology, Ruhr University Bochum, Bochum, Germany. Summary Small heat shock proteins (sHsps) including the wellstudied IbpA protein from Escherichia coli are molecular chaperones that bind to non-native proteins and prevent them from aggregation. We discovered an entirely unexplored tricistronic small heat shock gene cluster in Pseudomonas putida. The genes pp3314, pp3313 and pp3312 (renamed to hspX, hspY and hspZ respectively) are transcribed in a single transcript. In addition to σ32-dependent transcriptional control, translation of the first and second gene of the operon is controlled by RNA thermometers with novel architectures. Biochemical analysis of HspY, HspZ and P. putida IbpA demonstrated that they assemble into homo-oligomers of different sizes whose quaternary structures alter in a temperature-dependent manner. IbpA and HspY are able to prevent the model substrate citrate synthase from thermal aggregation in vitro. Increased stress sensitivity of a P. putida strain lacking HspX, HspY and HspZ revealed an important role of these sHsps in stress adaptation. The hspXYZ operon is conserved among metabolically related bacteria that live in hostile environments including polluted soils. This heat shock operon might act as a protective system to promote survival in such ecological niches. Introduction Small heat shock proteins (sHsps) are a widely distributed group of proteins in all domains of life (Narberhaus, 2002). In eukaryotes, sHsps confer tolerance to stress conditions and sHsp dysfunction can cause a range of human

Received 2 August, 2013; accepted 13 February, 2014. *For correspondence. E-mail [email protected]; Tel. +49 (0) 234 322 3100; Fax +49 (0) 234 321 4620.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd

pathologies (Sun and MacRae, 2005). Bacterial sHsps are an integral part of a universal stress protection system, the so-called heat shock response (Yura et al., 2000; Lim and Gross, 2011). In response to protein denaturation, bacteria synthesize a variety of heat shock proteins (Hsps) including proteases and chaperones (Gross, 1996; Bukau and Horwich, 1998; Yura et al., 2000). The role of sHsps in this multichaperone network is to bind to non-native and unfolded proteins and to maintain them in a refolding-competent state (Narberhaus, 2002). Client proteins bound to sHsps are prevented from irreversible aggregation and can be refolded by the other chaperones (Veinger et al., 1998; Mogk et al., 2003). By this ATP-independent ‘holdase’ function, sHsps prevent an overload of the major chaperone machineries, GroEL/ GroES and DnaK/DnaJ/GrpE, during heat stress conditions and serve as a reservoir of partially unfolded proteins (Ehrnsperger et al., 1997; Lee et al., 1997; Veinger et al., 1998). Most sHsps share a conserved central domain of ∼ 90 amino acids (Caspers et al., 1995). Due to sequence homology with the vertebrate eye lens protein α-crystallin, this domain is referred to as α-crystallin domain. It is preceded by an N-terminal region and followed by a short C-terminal tail that both can differ in length and sequence. As their name already indicates, sHsps are characterized by a low molecular mass of 12 to 43 kDa with an average weight between 14 kDa and 27 kDa. Most of the sHsps assemble into large oligomeric complexes with molecular masses of 800 kDa or even higher (Narberhaus, 2002). In most Gram-negative bacteria, transcription of small heat shock genes is regulated by the alternative sigma factor σ32 (RpoH) (Yura et al., 1993). Translational upregulation of σ32 protein combined with a temporal stabilization under protein-denaturing conditions result in increased amounts of σ32 and subsequent expression of multiple heat shock genes (Bukau, 1993; Gross, 1996; Yura et al., 2000). Transcription of Pseudomonas putida rpoH is in part controlled by the extracytoplasmic function sigma factor σE that couples the membrane status to the amount of σ32 (Aramaki et al., 2001; Alba and Gross, 2004).

1836 S. S. Krajewski, M. Joswig, M. Nagel and F. Narberhaus Additionally, expression of sHsp genes can be regulated on the translational level by cis-regulatory RNA elements that respond to temperature (Kortmann and Narberhaus, 2012). These so-called RNA thermometers (RNATs) are mainly located in the 5′ untranslated region (5’UTR) of a messenger RNA (mRNA). Under low temperature conditions, they fold into a secondary structure that sequesters the ribosome binding site (ShineDalgarno sequence, SD) and thus prevent translation initiation. An increase in temperature results in the destabilization of the RNA structure that in turn leads to liberation of the SD sequence and translation initiation. Dual control was also shown for the ibpA gene (inclusion body-associated protein A) in the soil bacterium Pseudomonas putida. Here, synthesis of IbpA is regulated on the transcriptional level by σ32 and on the translational level via a minimalistic ROSE-like (Repression Of heat Shock gene Expression) RNAT (Krajewski et al., 2013). The major characteristic of ROSE-like elements is an anti-SD sequence composed of a U(U/C)GCU motif (Chowdhury et al., 2003; 2006; Waldminghaus et al., 2005). Translational regulation of ibpA by this RNAT is conserved between various Pseudomonas species. In contrast to sHsps in many other bacterial species, IbpA is critically important for fitness of P. putida under heat stress conditions. An ibpA deletion mutant exhibits a severe growth defect under heat stress conditions and is delayed in heat stress recovery (Krajewski et al., 2013). Besides heat stress, P. putida frequently encounters a wide variety of stress conditions since it is able to inhabit diverse ecological niches including polluted and inhospitable environments (Timmis, 2002). By a bioinformatic approach, we found three consecutive genes, pp3314, pp3313 and pp3312, which encode novel putative sHsps. As these proteins share only limited similarity to IbpA-like sHsps, which are often called HspA, HspB, HspC, etc. (Narberhaus, 2002), we named them HspX, HspY and HspZ (Supporting Information Fig. S1). Here we report the regulation, protein characteristics and physiological role of these proteins in P. putida and provide evidence that they exist in many environmental bacteria. Results Transcriptional control of the hspXYZ genes As the genes hspXYZ encode putative small heat shock proteins, we first analysed their expression under heat stress conditions. P. putida cells were grown to exponential phase at 25°C and heat shocked to 42°C for 2 h. Northern blot analyses were performed with specific DNA probes against the genes hspX, hspY and hspZ (Fig. 1A) and revealed heat-dependent increase of transcription (Fig. 1B–D). Each probe detected a transcript with a length of about 1500 nucleotides (nt) suggesting that the

hspXYZ genes are organized in a tricistronic operon. Synthesis of the mRNA peaked around 15 min after heat shock. Using specific HspY antisera, we found a slightly delayed production of the protein with a maximum 30 min after temperature upshift (Fig. 1E). Significant amounts of HspY protein were detectable even 120 min after heat treatment. To study the regulation of the operon, the 5′ end of the P. putida hspXYZ transcript was determined by 5′ RACE. Total RNA isolated from heat-shocked cells was either untreated or treated with tobacco acid pyrophosphatase (TAP), which is specific for triphosphorylated 5′ ends. This treatment results in enhancement of primary transcripts because monophosphorylated ends generated by this treatment are suitable for adapter ligation. 5′ RACE-PCR gave rise to a prominent band, which exclusively appeared after TAP treatment (Fig. 2A). Sequencing of the fragment revealed a 5′ end at position −72 with respect to the first nucleotide of the translational start codon (Fig. 2B). The promoter region upstream of the transcriptional start site (+1) shares high similarity with the −10 and −35 consensus sequence (NNCTTGAA-N(1318)-CNCCATAT) of an Escherichia coli σ32-type promoter (Wade et al., 2006) suggesting σ32-dependent transcriptional regulation of hspXYZ. To confirm that this operon belongs to the σ32 regulon, we analysed the expression of plasmid-encoded P. putida hspXYZ in E. coli ΔrpoH lacking σ32 and the parental E. coli wild type (wt; Fig. 2C). A plasmid harbouring the entire hspXYZ operon including its promoter region was transferred into both strains. Low amounts of hspXYZ transcript in the E. coli wt strain at 25°C significantly increased after heat shock to 37°C. The transcript of 1500 nt provides additional evidence for hspX, hspY and hspZ being located on a single transcription unit. In contrast, the hspXYZ transcript was absent in the ΔrpoH strain at both temperatures demonstrating transcriptional control by the heat shock sigma factor σ32. The hspXYZ operon is differentially regulated at the posttranscriptional level by two distinct RNA thermometers Many IbpA-encoding heat shock genes are controlled on the translational level by ROSE-like RNATs (Waldminghaus et al., 2005; Krajewski et al., 2013). To examine temperature-mediated translational control of hspXYZ, we used a well-established E. coli reporter gene system (Klinkert et al., 2012). The 5’UTRs were translationally fused to the reporter gene bgaB which codes for a heat stable β-galactosidase (Hirata et al., 1986). In this system, the 5’UTR-bgaB fusions are under control of the arabinose-inducible promoter, which allows temperature-independent transcriptional control (Guzman

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1835–1853

Novel small heat shock proteins in Pseudomonas putida

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Fig. 1. Transcriptional and translational regulation of the P. putida hspXYZ operon. A. Genetic organization of the hspX, hspY and hspZ genes and their flanking region. Locations of DNA probes used in B–D are indicated by grey bars. B–E. Heat induction of the hspXYZ transcript. P. putida cells were grown to OD600 0.5 at 25°C and heat shocked at 42°C. Samples were taken at the indicated time points after the temperature upshift and probed by Northern (B–D) and Western blot analyses (E). Northern analyses were performed with probes against hspX (B), hspY (C) and hspZ (D) transcripts. Upper panel: Northern blot; lower panel: ethidiumbromide-stained 16S rRNA fractions were used as loading control. E. Western analysis was performed with specific α-HspY antisera.

Fig. 2. Transcriptional start site determination and σ32-dependent transcription of the hspXYZ operon. A. The 5′ end of the P. putida hspXYZ transcript was determined by 5′ RACE. Experiments were performed with RNA extracted from heat-shocked P. putida cells (10 min at 42°C). RNA was treated with (+) or without (−) TAP prior to adapter ligation and reverse transcription. The ethidiumbromide-stained agarose gel of PCR products obtained with 5′ RACE is shown. The fragment that was eluted and sequenced is marked by an asterisk. Control PCR was performed with total RNA used for the 5′ RACE and chromosomal DNA as template. bp: basepairs. B. Comparison of the hspXYZ promoter region with the consensus sequence of an E. coli σ32 promoter (Wade et al., 2006). The −35 and −10 regions are marked in grey. The determined 5′ end (5′ RACE; A) of the mRNA (+1) is marked by an arrow. nt: nucleotides. C. Verification of σ32 (RpoH)-dependent regulation of the P. putida hspXYZ operon. A plasmid harbouring the P. putida hspXYZ operon including its promoter region was transferred into E. coli ΔrpoH and the parental wild type (wt) strain. Both strains were grown at 25°C to OD600 0.5 and heat shocked at 37°C. Samples for RNA isolation were taken 10 min after stress induction and analysed via Northern blot analysis (hspY probe; upper panel). Ethidiumbromide-stained 16S rRNA served as loading control (lower panel).


1838 S. S. Krajewski, M. Joswig, M. Nagel and F. Narberhaus

Fig. 3. Translational control of the hspXYZ operon. A. Schematic view of translational reporter gene fusions used in this study. Regions spanning from the transcriptional start site to the AUG including one triplet of the coding region of hspX, hspY and hspZ were translationally fused to bgaB. B. Temperature-dependent expression of the hsp-bgaB fusions. β-galactosidase assays were carried out with the translational reporter gene fusions (shown in A) in E. coli DH5α. Cells harbouring the corresponding plasmids were grown at 25°C to OD600 0.5 prior to induction of transcription with 0.01 % L-arabinose and transfer of part of the culture to 42°C. β-galactosidase activity (in Miller units, MU) was measured 30 min after stress induction. The average results of three independent measurements are shown with indicated standard deviations. Induction factors are given above the columns.

et al., 1995). hspX, hspY and hspZ regions spanning from the transcriptional start site to the AUG start codon plus one triplet of each open reading frame were fused to bgaB (Fig. 3A). At 25°C, the hspX fusion allowed basal β-galactosidase activity of 10 Miller units (MU) that increased 10.5-fold after a temperature upshift to 42°C suggesting the presence of a functional RNAT (Fig. 3B). A comparable induction of reporter gene activity (8.8-fold) was observed for the hspY fusion suggesting either a polar effect of the hspX RNAT on hspY expression or an independent intergenic RNAT upstream of hspY. In con-

trast, the hspZ-bgaB fusion exhibited low reporter gene activity and minor heat induction (2.8-fold). As the thermo-regulated hspY fusion also encodes the hspX gene, we tested whether the HspX protein affects expression of the hspY gene. The hspX AUG start codon was exchanged for a stop codon to prevent HspX synthesis. The hspY ΔhspX fusion exhibited reduced reporter gene activity at both temperatures. However, an 8.6-fold temperature-dependent induction indicated that thermometer instead of thermom-eter function was retained. To identify the region important for RNAT function in more


Novel small heat shock proteins in Pseudomonas putida detail, we used 200 nt and 100 nt upstream of hspY as well as the intergenic region between hspX and hspY (IGR; 27 nt) and examined temperature-dependent translational control (Fig. 3A). For all three versions, reporter gene activity significantly increased when cultures were shifted from 25 to 42°C (Fig. 3B). The intergenic fusion resulted in overall elevated β-galactosidase activity at low and high temperature. Increased reporter gene activity at 25°C might indicate a less stable secondary structure and less effective inhibition suggesting that nucleotides in the coding region of hspX are needed to form an inhibitory structure. Taken together, the hspX and the hspY gene but not hspZ are temperature-dependently regulated on the translational level by RNATs. Analysis of the RNAT structures and heat-induced melting To gain further insights into the mechanism of translational control of hspX and hspY, we performed structure probing experiments. In vitro transcribed and radioactively labelled RNAs were treated with RNase T1 (cuts single stranded guanines) and nuclease S1 (cuts singlestranded nucleotides) at 25 C and 42°C prior to gel electrophoresis. The hspX 5’UTR is predicted to form a secondary structure consisting of two hairpins sequestering the SD region and the AUG start codon in the 3′ proximal hairpin II. The overall cleavage pattern (Fig. 4A) of the hspX 5’UTR obtained with RNase T1 and nuclease S1 at low temperature are fully consistent with the predicted secondary structure (Fig. 4B). RNase T1 and nuclease S1 cleavage at 25°C around position G18 confirmed the terminal loop of hairpin I. Conversely, protection of the nucleotides at position 7–16 and 23–35 supported formation of the stem region of hairpin I. The stem region of hairpin II was verified by the absence of RNase T1 and nuclease S1 cleavage around position G51 and the SD region (around position G66), whereas susceptibility of region 58–61 for RNase T1 and nuclease S1 cleavage supported the terminal loop of hairpin II. Formation of hairpin I was unaffected by temperature as its nucleotides remained protected against cleavage at 42°C, a possible consequence of many stable G-C pairs. In contrast, the guanines of the SD (G62, G63, G65 and G66) and anti-SD region (G51 and G55) became susceptible to RNase T1 cleavage at 42°C demonstrating melting of hairpin II and liberation of the ribosome binding site (Fig. 4A, B). Nuclease S1 cleavage of this region exclusively at 42°C supports the results obtained from RNase T1 treatment. As 100 nt upstream of the hspY gene were sufficient for thermoregulation in the reporter gene studies, we used this region for structural analysis. The predicted two-

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hairpin structure occludes the SD sequence in hairpin II (Fig. 4D). Around position 43 (marked with an asterisk in Fig. 4C) the nucleotide ladder was compressed making it difficult to precisely assign individual nucleotides in stem I. Nuclease S1 cleavage revealed a susceptible region, which is cleaved even at 25°C. The flanking regions are resistant to S1 and T1 cleavage at 25 C and 42°C supporting the predicted long helical structure between positions 1–52 (Fig. 4D), in which the guanines are paired and thus would hamper RNase T1 cleavage even at 42°C. T1 and S1 cuts in the region between 58 and 62 were consistent with a linker region between both hairpins. Cleavage of nucleotides around G76, G81 and G82 by RNase T1 and nuclease S1 supported the terminal loop of hairpin II. Protection of the nucleotides around position 67 from nuclease S1 cleavage and only marginal cleavage of the SD guanines by RNase T1 at 25°C support the hairpin II stem region. Accessibility of the SD region (G86, G89, G90, G92 and G93) was significantly increased at 42°C providing evidence for temperature-induced loosening of hairpin II accompanied by liberation of the SD region. Notably, several Mg2+-independent strand breaks occurred whose intensity increased over time in hairpin II suggesting that this region is rather flexible (lane C; Fig 4C and data not shown). The calculated minimum free energies (ΔG) of the hspX and hspY RNATs (−21.10 and −27.00 kcal mol−1 respectively) are comparable to other known RNATs (Kortmann and Narberhaus, 2012). Collectively, these results support the model that the hspX 5’UTR and the upstream region of hspY fold into secondary structures that alter in a temperature-responsive manner. Notably, the structures of the hspX and hspY RNATs differ from each other and from all previously described RNATs (Kortmann and Narberhaus, 2012). The composition of homo-oligomeric complexes of IbpA, HspY and HspZ is temperature dependent To analyse the protein characteristics of the P. putida sHsps, we produced the proteins HspX, HspY, HspZ and IbpA both with and without a carboxy-terminal hexahistidinetag (His6) using the pET-expression system. The molecular mass of the untagged proteins was calculated from the amino acid sequences and were HspX: 16 889 Da, HspY: 13 993 Da, HspZ: 19 209 Da and IbpA: 16 349 Da. His6-tagged proteins are expected to have 823 Da higher molecular masses. HspX was only synthesized in small amounts and was insoluble precluding its biochemical characterization (Supporting Information Fig. S2). In contrast, His6-tagged and untagged HspY, HspZ and IbpA were produced in high amounts, and a major fraction of the proteins was soluble (Fig. 5A upper panel and data not shown).



Fig. 4. Secondary structure analysis and temperature-dependent alterations of the hspX and hspY RNATs. A. Structural analysis of the hspX 5′UTR. Enzymatic cleavage of 5′ end-labelled RNA was performed with RNase T1 (0.002 U) and nuclease S1 (0.17 U) at 25 and 42°C. RNA fragments were separated on an 8% polyacrylamide gel. Lane C, RNA treated with water instead of RNase served as control; lane T, RNase T1 cleavage at 50°C under denaturing conditions; lane L, alkaline ladder. B. Structure model of the hspX RNAT with probing results of RNase T1 treatment. Cleavage sites introduced by RNase T1 at 25 and 42°C are indicated by arrows and arrows with asterisks respectively. Nucleotides of the SD region and the AUG start codon are depicted in bold letters. Thermodynamical stability (ΔG) is given below the structure. C. Structural analysis of the intergenic hspX RNAT. The upstream region (100 nt) of the hspY gene was structurally analysed as described in A. D. Structure model of the intergenic hspY RNAT with probing results of RNase T1 treatment. 100 nt upstream of the hspY gene were used for secondary structure prediction as described in B. asterisk: compressed nucleotide ladder. Nucleotides of the stop codon of hspX and of the SD region and AUG start codon of hspY are depicted in bold letters.



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Fig. 5. sHsp purification and oligomer formation. A. Heterologous production and purification of sHsps. C-terminally His6-tagged and untagged versions of IbpA, HspZ and HspY were heterologously expressed in E. coli BL21(DE3). Escherichia coli cells harbouring the expression plasmids were grown at 37°C to an OD600 of 0.5. Proteins were synthesized for 2 h after addition of 1 mM IPTG. Upper panel: SDS-PAGE of crude extracts containing synthesized sHsps; lower panel: His6- and untagged sHsps were subjected to Ni-NTA chromatography. Elution fractions were separated by SDS-PAGE. Molecular mass markers (in kDa) are given on the left of the gels. B. Co-purification procedure to analyse complex formation. Crude extract containing untagged sHsp was incubated with a crude extract containing His6-tagged sHsp. The mixture was applied to small scale Ni-NTA affinity chromatography (see experimental procedures). Elution fractions were analysed by SDS-PAGE. C. Co-purification of various sHsp combinations. Elution fractions after co-purification were separated by SDS-PAGE. Protein bands are marked by asterisks. Molecular mass markers (in kDa) are indicated on the left of the gel. D. Summary of the co-purification analyses performed with the sHsp in various combinations. +: stable interaction; +/−: weak interaction; -: no interaction.

Since many sHsps assemble into oligomeric complexes, we investigated the interaction between IbpA, HspY and HspZ using a co-affinity purification approach. For this purpose, crude extracts containing His6-tagged and untagged sHsp were mixed prior to Ni-NTA purification (Fig. 5B). Purification of each His6-tagged protein individually resulted in a single band, whereas untagged proteins were not retained on the column (Fig. 5A). In case of oligomer formation between His6-tagged and untagged sHsp, the co-purified untagged protein should give rise to a second band on SDS gels. First, we tested the homo-oligomer formation of sHsps by incubating each His6-tagged protein with its untagged version. The appearance of two bands after purification demonstrated that IbpA, HspZ and HspY assemble into homo-oligomers (Fig. 5C, D). Formation of hetero-oligomeric complexes was analysed by combining different sHsps. Low to barely visible amounts of co-purified sHsp suggested less stable or only transiently formed hetero-oligomeric complexes. Using alternative protocols, e.g. denaturation/ renaturation as in Studer and Narberhaus (2000), also did not provide evidence for hetero-oligomer formation (data not shown). To determine homo-oligomer complex sizes, we performed size exclusion chromatography (SEC) with purified IbpA-His6, HspY-His6 and HspZ-His6. HspY-His6

assembled into complexes with molecular masses of 55 and 16.5 kDa representing tetramers and monomers respectively (Fig. 6A). Formation of distinct homooligomeric complexes was also shown by native PAGE (Fig. 6C). The small HspY-His6 complexes disappeared at 43°C suggesting assembly of these complexes into high molecular weight complexes. HspZ-His6 formed homooligomers composed of six up to roughly 60 subunits that exhibit molecular masses of 125 kDa up to 1300 kDa (Fig. 6B). The broad peaks suggest a polydisperse nature of these complexes. At low temperatures [ice and room temperature (RT)] HspZ-His6 seems to favour the smaller complex size, whereas at 43°C the equilibrium shifted towards complexes with higher molecular mass giving rise to a second band in native PAGE (Fig. 6C). IbpA-His6 appeared in the void volume (data not shown) in agreement with native PAGE analysis revealing large oligomeric complexes at low temperature, which did not enter the gel (Fig. 6C). A distinct band at RT and at 43°C suggests the dissociation of the high molecular weight complexes into complexes with defined sHsp subunits numbers. In summary, IbpA-His6, HspY-His6 and HspZ-His6 form different temperature-regulated homo-oligomeric complexes, which might explain why they do not efficiently form heteromeric complexes.


1842 S. S. Krajewski, M. Joswig, M. Nagel and F. Narberhaus Fig. 6. Temperature-dependence of homooligomeric sHsp complexes. Size exclusion chromatography was performed with purified (A) HspY-His6 and (B) HspZ-His6 with a Superdex 200 10/300 GL column. Five hundred microlitre fractions were collected and analysed by SDS-PAGE. Complex sizes are indicated at the corresponding peaks on the elution profile determined by calibration of the column with high molecular mass standards. C: control, aliquot of protein extract loaded on the column. C. Temperature-dependent homo-oligomer formation. 7.5 μg of HspZ-His6, HspY-His6 and IbpA-His6 were incubated on ice, at RT or at 43°C prior to native PAGE.

HspX and IbpA but not HspZ exhibit typical chaperone activity The key characteristic of sHsps is to bind denatured proteins and hold them in a refolding-competent state (Narberhaus, 2002). To assess the chaperone activity of IbpA, HspY and HspZ, we tested their ability to protect the model substrate citrate synthase (CS) from aggregation at elevated temperatures. As positive and negative control, we used bovine α-crystallin and BSA respectively. α-Crystallin and P. putida IbpA were equally efficient molecular chaperones as they suppressed CS aggregation in a concentration-dependent manner (Fig. 7A and C). HspY exhibited a reduced ability to prevent heat-

induced CS aggregation (Fig. 7D). Like the negative control BSA, HspZ was not capable to protect CS from thermal aggregation in vitro (Fig. 7B and E). A possible cooperation of HspZ and HspY in chaperone activity was excluded as a combination of both proteins had no positive influence on CS protection mediated by HspY (Fig. 7F). We conclude that IbpA and HspY are typical sHsps with characteristic chaperone activity, whereas HspZ may have a more specific role in P. putida. sHsps are important for stress management of P. putida Phenotypical analyses of a P. putida strain lacking IbpA revealed its importance for fitness under heat stress



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Fig. 7. Chaperone activity of P. putida sHsps. Thermally induced aggregation of CS (900 nM) at 43°C is depicted as a function of time in the presence of various amounts of bovine α-crystallin (A), BSA (B), IbpA-His6 (C), HspY-His6 (D), HspZ-His6 (E) and a combination of HspY-His6 and HspZ-His6 (F). Aggregation was monitored by the increase in absorbance at 320 nm and normalized. Citrate synthase was incubated in the absence (ratio 1:0) and the presence of bovine α-crystallin, BSA and sHsps at final concentration of 225 nM (1:0.25), 450 nM (1:0.5), 900 nM (1:1) and 1.8 mM (1:2). The absorbance of all tested sHsps in the absence of CS was monitored (0:1).

condition (Krajewski et al., 2013). To determine the physiological role and relevance of HspX/Y/Z, we deleted the hspXYZ operon in the P. putida wt and the ΔibpA strain. All strains grew equally well in liquid media when grown at 25°C or after heat shock to 37°C (Fig. 8A and B). However, all three mutants showed a severe growth defect when they were challenged with heat stress at 42°C (Fig. 8C). Three hours after temperature upshift, the cell density of the mutant cultures was approximately 40–50% lower as compared with the wt culture. Consistent with the heat-sensitive phenotype in liquid media, growth of the ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ mutants was reduced under continuous heat stress at 40°C on solid media (Fig. 8E). Heat-sensitivity of the ΔibpA and ΔhspXYZ strain was indistinguishable suggesting that the chaperone systems can partly compensate for each other. Heat sensitivity was exacerbated in the quadruple mutant (Fig. 8C and E). Since P. putida has the ability to inhabit environments contaminated with aromatic substances, we asked whether HspX, Y and Z are involved in tolerance against the toxic effects of aromatic compounds. All mutants showed a mild but reproducible sensitivity to phenol compared with the wt on solid media and in liquid media at 25°C (Fig. 8F and G), which is more pronounced at 37°C

when lower phenol concentrations are sufficient to inhibit growth (Fig. 8H). To further examine the phenol sensitivity of the mutant strains, growth competition experiments were performed between the P. putida wt and the ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ mutant strains. Equal amounts of wt and mutant cells were mixed and co-cultured for 25 h either with or without phenol at 25°C. The number of colony forming units of competing cells was determined (ratio in %) before and after 25 h incubation. Incubation at 25°C without phenol let to a slight decrease of the mutant cells (Fig. 8I). Under phenol stress, growth of the mutant strains was severely compromised, decreasing from 23 to 10%, 28 to 15% and 29 to 4% for the ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ strains respectively (Fig. 8I). Discussion The hspXYZ operon is differentially regulated by two RNATs with novel architectures The P. putida hspXYZ operon has several remarkable features. Like the ibpA gene (Krajewski et al., 2013), it is transcribed from a σ32-dependent promoter in response to protein denaturing conditions like heat shock (Fig. 1) or urea treatment (data not shown). Also like ibpA,



Fig. 8. Stress tolerance of the P. putida wild type, ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ. A–C. Bacterial growth under optimal growth conditions and elevated temperatures in liquid media. P. putida wild type (wt), ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ cells were grown at 25°C to an OD600 of 0.5 (black arrow) and grown at 25°C (A), 37°C (B) or 42°C (C). OD600 was monitored. D–F. Growth of P. putida wt, ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ at different stress conditions on agar plates. Cells were grown to OD600 of 0.5 at 25°C and serially diluted 10-fold. Three microlitre of each suspension was spotted onto LB solid media (D, E) and LB media containing 10 mM phenol (F). Plates were incubated at 25°C (D, F) or 40°C (E). G–H. Pseudomonas putida wt, ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ challenged with solvent stress. P. putida wt, ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ were grown to an OD600 of 0.5 and incubated with 16 mM phenol at 25°C (G) and 10 mM phenol at 37°C (H). OD600 was monitored. I. Growth competition assay. Equal cell amounts of P. putida wild type, ΔibpA, ΔhspXYZ and ΔibpAΔhspXYZ cells were mixed. The fractions of each strain was determined (ratio in %) before and 25 h after growth at 25°C in the absence (−) or presence (+) of 16 mM phenol as described in ‘Experimental procedures’.

translation of the hspXYZ operon is under RNAT control (Fig. 9). The mechanism of translational regulation, however, is unique because two structurally different and not yet described RNATs modulate translation of the first two genes of this operon. The 5’UTR of hspX folds into a two-hairpin structure. The first hairpin is composed of many G-C base pairs providing high stability and thus preventing melting of this hairpin even at heat stress temperature. Such 5′ proximal stable stem-loop structures are thought to increase transcript stability (Emory et al., 1992). The second hairpin formed in the hspX 5’UTR

engages the SD sequence and the start codon. As in other two-hairpin RNATs, the 3′ proximal hairpin of the hspX-UTR serves as temperature-responsive element that melts in a temperature-dependent manner (Waldminghaus et al., 2007; Krajewski et al., 2013). Remarkably, its sequence and architecture differs from all known RNAT structures. RNATs typically harbour destabilizing elements, like mismatches and loops, and stabilizing elements such as G-C pairs (Kortmann and Narberhaus, 2012). A delicate balance of these elements is crucial for sufficient repression at low temperature and



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Fig. 9. Regulation and function of P. putida hspXYZ and ibpA. Expression of the tricistronic hspXYZ operon and the monocistronic ibpA gene is transcriptionally controlled by the heat shock sigma factor σ32. Translation of ibpA, hspX and hspY is temperature dependently regulated by distinct RNATs. Insolubility of the HspX prevented its biochemical analysis. HspY, HspZ and IbpA assemble into homo-oligomeric complexes, whose quaternary structure alter in a temperature-dependent manner. HspY and IbpA, but not HspZ, exhibit CS chaperone activity in vitro.

melting at higher temperature (Rinnenthal et al., 2011). Melting of the hspX RNAT in the appropriate temperature range may be facilitated by the two internal loops (destabilizing) and four G-C base pairs (stabilizing; Fig. 4). The vast majority of RNATs are located at the 5′ end of mRNAs (Kortmann and Narberhaus, 2012). Only one RNAT located in the intergenic region of a bicistronic operon has been described so far (Böhme et al., 2012). The intergenic region (124 nt) of the Yersinia pseudotuberculosis yscW-lcrF transcript folds into a secondary structure composed of two hairpins. This in-build thermoregulatory element is important for fine-tuned regulation of LcrF, a master regulator of Yersinia virulence. As the SD sequence of lcrF is masked by a consecutive stretch of four uridines, this RNAT is a member of the fourU family. Here, we describe the second example of an intercistronic RNAT upstream of the hspY gene of the P. putida hspXYZ operon. Although the intergenic region, composed of only 27 nt, was able to confer thermoregulation in vivo, repression at low temperature was more effective with longer fragments suggesting the involvement of nucleotides in the hspX coding region in thermoregulation. A fusion consisting of 100 nt upstream of the hspY start codon was sufficient for full RNAT function in vivo. Like the hspX RNAT, this novel intergenic RNAT harbours an internal loop and stem regions containing

G-C pairs that are probably important for thermoregulation (Fig. 4). The presence of structurally different RNATs upstream of two subsequent open reading frames in a single transcription unit is contrasted by a recently reported situation in which different RNATs are located on three separate transcripts of Neisseria meningitidis. All three RNATs team up to induce expression at 37°C of genes involved in immune evasion (Loh et al., 2013). These recent findings on the modular nature of RNATs suggest that many more might be found in previously unexpected positions in polycistronic transcripts (Narberhaus, 2013). The sHsps IbpA, HspY and HspZ differ in quaternary structure and chaperone activity The intricate regulation of the hspXYZ operon suggested important cellular functions of the encoded proteins, which prompted us to study their biochemical properties. Pseudomonas putida IbpA is most similar to E. coli IbpB not only at sequence level but also according to its chaperone activity and complex formation (Shearstone and Baneyx, 1999). HspY and HspZ differ substantially from IbpA both in sequence and quaternary structure. sHsp dimers, assembled via the α-crystallin domain, usually are the basic building blocks of higher molecular weight complexes (Kim et al., 1998; van Montfort et al., 2001). Beside the α-crystallin domain, the highly variable N- and


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S. S. Krajewski, M. Joswig, M. Nagel and F. Narberhaus

C-termini are most likely determinants for oligomer formation (Shearstone and Baneyx, 1999; Narberhaus, 2002; Nakamoto and Vígh, 2007). A conserved Isoleucin-XIsoleucin/Valin (IXl/V) motif, with X representing any amino acid, in the C-terminal extension presumably pairs with a hydrophobic patch in the α-crystallin domain of the neighbouring sHsp (Studer et al., 2002; Pasta et al., 2004). Likewise, the C-terminal extensions of HspX, HspY and HspZ harbor the IXI/V motif suggesting the same mechanism to form and stabilize oligomers (Supporting Information Fig. S1). Although HspX, HspY and HspZ share sequence homology, both termini, especially the N-termini, differ in length and sequence, already indicating different oligomerization behaviours. HspY predominantly forms small homo-oligomeric complexes that assemble into high molecular weight complexes after a temperature upshift (Fig. 9). HspZ forms homo-oligomers of two main populations with a preference for higher multimeric complexes at high temperature (Fig. 9). sHps from different classes are unable to associate (Studer and Narberhaus, 2000) and the differences between IbpA, HspY and HspZ are probably responsible for the observed weak hetero-oligomer formation. sHsp oligomers are dynamic structures whose subunits constantly exchange (Bova et al., 2000; Studer and Narberhaus, 2000; Sobott et al., 2002; Lentze et al., 2004), and temperature-dependent structural changes are proposed to play a key role in sHsp action and chaperone activity (Lee et al., 1997). Structural alterations finally result in exposure of hydrophobic regions (Shearstone and Baneyx, 1999; Yang et al., 1999; Giese and Vierling, 2002; Nakamoto and Vígh, 2007; Franzmann et al., 2008). It is difficult to distinguish between protein regions that are necessary for chaperone activity and for oligomerization as both are based on hydrophobic interactions. Although sHsps bind a variety of non-native proteins in vitro and protect a wide range of proteins in vivo (Lee et al., 1997; Studer and Narberhaus, 2000; Basha et al., 2004b), this does not necessarily exclude a preference for certain substrates. Substrate protein profiles of two S. cerevisiaes Hsps from the same compartment overlap but are not identical demonstrating at least some selectivity of sHsps for their substrates (Haslbeck et al., 2004). Agrobacterium tumefaciens HspL predominantly stabilizes the VirB8 protein of the type IV secretion system (Tsai et al., 2010; 2012). Despite such selected examples, substrate specificity of sHsps in vivo is largely unknown (Leroux et al., 1997; Kokke et al., 1998; Basha et al., 2012). As the α-crystallin domain is conserved, most likely variable sequences outside of the domain are involved in substrate selection. Several sites within the C- and N-terminus are postulated to be important for chaperone function or rather substrate recognition (Lindner et al., 2000; Basha et al., 2004a; Stromer et al.,

2004). The diverse N-termini of HspX, Y and Z (Supporting Information Fig. S1) might be indicative of different substrate specificity. Moreover, low in vitro chaperone activity of HspY and the inability of HspZ to protect CS from thermal aggregation might reflect specialized substrate specificity or alternative functions in vivo. Apart from their classical chaperone function, sHsps are involved in membrane maintenance (Nakamoto and Vígh, 2007). Stresses, like sudden temperature upshifts, have a severe impact on membrane integrity. In Gramnegative bacteria, such changes indirectly activate the σE regulon and in turn induce the heat shock response including sHsp synthesis. Hsp17 of the cyanobacterium Synechocystis specifically interacts with lipids of the thylakoid membrane and stabilizes it under heat shock conditions (Török et al., 2001). Hence, a role of HspX, Y and Z in membrane maintenance is conceivable, in particular as P. putida is often exposed to membranedamaging agents in its natural environment. The hspXYZ operon is only found in metabolically related environmental strains After discovery of the hspXYZ operon, we found many related sequences in other environmental bacteria (Supporting Information Fig. S3). The identity of HspX, Y and Z protein sequences ranges from 30 to 63%. Remarkably, all proteins encoded by the first gene in these operons cluster together in one branch together with HspX (Fig. 10). A similar clustering was observed for the HspY and HspZ homologues encoded by the second and the third gene respectively. All three branches are clearly separated from the IbpA-like proteins from E. coli, P. putida and P. aeruginosa. In some cases, we found reduced operons encoding the HspX and HspY homologues only (marked with a hash key in Fig. 10) or single genes coding for HspZ homologues (marked with an asterisk in Fig. 10), for instance the bicistronic mpe_ A2043/mpe_A2042 region from Methylibium petroleiphilum or the monocistronic ctgr7-1921 gene from Thioalkalivibrio sulfidophilus. It is striking that hspXYZ homologues were only found in environmental bacteria. Except for Anaerolinea thermophila (phylum: Chloroflexi), these bacteria belong to different classes of the largest and phenotypically most diverse phylum proteobacteria. One remarkable feature of almost all of these bacteria is their high metabolic versatility. Pseudomonas putida has exceptional metabolic capabilities in degrading a wide variety of natural and man-made aromatic compounds (Nelson et al., 2002; Timmis, 2002). The beta-proteobacteria Rubrivivax benzoatilyticus and M. petroleiphilum PM1 metabolize a wide range of aromatic hydrocarbons among them are benzoate, phthalate and cyclohexanol or toluene, phenol



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Fig. 10. HspX, HspY and HspZ homologs in metabolically related bacteria. Phylogenetic tree of HspX, HspY and HspZ homologues and IbpA-like proteins based on ClustalW alignment of the deduced amino acid sequences derived from public databases. The HspX/Y/Z homologues originate from the following bacteria: Pseudomonas putida KT2440, Rubrivivaxbenzoatilyticus JA2, Geobacter sulfurreducens PCA, Acidovorax sp. CF316, Geobacter lovleyi SZ, Aromatoleum aromaticum EbN1, Methylococcus capsulatus str. Bath, Polymorphum gilvum SL003B-26A1, Methylobacterium extorquens AM1, Thioflavicoccus mobilis 8321, Anaerolinea thermophila UNI-1, Methylibium petroleiphilum PM1, Thioalkalivibrio sulfidophilus HL-EbGr7. IbpA homologues were obtained from E. coli, P. aeruginosa and P. putida. #: HspX or HspY-like proteins encoded in a bicistronic operon. *: monocistronically encoded HspZ-like protein.

and benzene (Nakatsu et al., 2006; Ramana Ch et al., 2006). Polymorphum gilvum SL003B-26A1 is even capable of using crude oil as the sole carbon and energy source (Nie et al., 2012). Aromatic compounds are not only potential nutrients for these bacteria but act as stressors due to their membrane-damaging effects. Because of their hydrophobicity, aromatic compounds preferentially dissolve in membranes (Sikkema et al., 1994). As a result the membrane fluidity increases, which in turn leads to a loss of ions, denaturation of membrane proteins and weakening of the proton motive force (Sikkema et al., 1995). In these ecologically niches, bacteria are confronted with a biodegradation-versus-stress dilemma (Carmona et al.,

2009). In accordance, P. putida primarily senses aromatic hydrocarbons like toluene and xylene as a stress trigger rather than as a carbon source (Domínguez-Cuevas et al., 2006). To overcome this dilemma, bacteria respond in two ways. On the one hand, the metabolism is adapted to the nutrient availability by expressing catabolic genes to degrade the cytotoxic compounds (Velázquez et al., 2006). On the other hand, proteins generally important for stress management are increasingly synthesized including members of the heat shock response. Succinate-utilizing Aromatoleum aromaticum induces several heat shock proteins, like HtpG and ClpB, when cells are shocked with aromatic solvents such as toluene and phenol (Trautwein et al., 2008). Interestingly,


1848 S. S. Krajewski, M. Joswig, M. Nagel and F. Narberhaus A. aromaticum also synthesized the HspX and HspY homologues Eba2730 and Ebb88, suggesting their contribution to cell protection under these condition (Trautwein et al., 2008). In agreement with that, we observed growth defects of P. putida strains lacking either IbpA or HspX, Y and Z under heat stress and solvent stress induced by phenol. We conclude that the novel heat shock operon hspXYZ is an integral part of a multifaceted chaperone network mediating stress tolerance in hostile ecological niches refractory to other microbes. Experimental procedures Bacterial growth conditions Bacterial strains used in this study are listed in Supporting Information Table S1. E. coli and P. putida strains were cultivated in Luria–Bertani (LB) medium at indicated temperatures. Media were supplemented with ampicillin (Ap, 150 μg ml−1), kanamycin (Km, 50 μg ml−1), nalidixic acid (Na, 300 μg ml−1), tetracycline (Tc, 10 μg ml−1) or gentamicin (Gm, 10 μg ml−1) if required.

Growth competition assay Pseudomonas putida wild type and mutant strains were grown to exponential growth phase in LB and adjusted to an OD600 of 0.1. Equal amounts of the cultures were mixed and incubated with or without 16 mM phenol (final concentration) at 25°C. Aliquots were taken before and 25 h after incubation. Samples were diluted 10−4, 10−5, 10−6, 10−7 and 10−8 depending on the OD600 of the culture and 200 μl of each serial dilution was plated in duplicate on LB plates without or with kanamycin (50 μg ml−1), gentamycin (12.5 μg ml−1) or both antibiotics. The numbers of colonies were counted after overnight incubation at 30°C and used to calculate the colonyforming units ratio of wild type and mutant strains.

Strain and vector constructions Oligonucleotides and plasmids used in this study are summarized in Supporting Information Tables S2 and S3. Recombinant DNA work was performed according to standard protocols (Sambrook and Russel, 2001). The nucleotide sequences of all constructs were confirmed by automated sequencing (Eurofins, Martinsried, Germany). For the construction of plasmid pBO1520 (promoter analysis) the P. putida hspXYZ gene region including its promoter region (140 bp upstream of hspX and 90 bp downstream of hspZ) was amplified and cloned into the singular SmaI site of pUC18. To construct translational bgaB fusions, the 5’UTRs were amplified by PCR and blunt-end subcloned into pUC18 (pBO2927, pBO2960, pBO2961). The bgaB fusions were cloned via primer derived NheI/EcoRI sites into the corresponding sites of pBAD2-bgaB (pBO2941, pBO2962, pBO2963). To obtain pBO2991, an exchange of the hspX AUG to a TAG in the hspY wild type fusion (pBO2960) was performed via site-directed mutagenesis with mutagenic

primers (listed in Supporting Information Table S2) according to the instruction manual of the QuikChange mutagenesis kit (Agilent Technologies, Santa Clara, USA). Translational bgaB fusions with 200 nt, 100 nt and 27 nt [hspXY intergenic region (IGR)] upstream of the hspY gene were constructed by insertion of NheI restriction sites in the hspY wild type fusion (pBO2960) via site-directed mutagenesis with mutagenic primers (listed in Supporting Information Table S2). Fragments from the transcriptional start site to the inserted restriction site were then deleted by digestion with NheI and re-ligation (plasmids pBO2989, pBO2990, pBO2992). To construct run-off plasmids used for in vitro transcription, the hspX 5’UTR or 100 nt upstream of hspY plus one triplet coding region were amplified with primers (Supporting Information Table S2), adding a T7 promoter sequence at the 5′ end and an EcoRV restriction site at 3′ end. Fragments were blunt-end cloned into SmaI site of pUC18 (pBO2993 and pBO2994). To construct plasmids for sHsp production, the ibpA, hspX, hspY and hspZ genes were amplified without its natural stop codon with primers that add an NdeI site at the 5′ end and a XhoI site, a stop codon and a SalI site at the 3′ end (listed in Supporting Information Table S2). These fragments were cloned into the NdeI/SalI site of pET24b to obtain the expression plasmids for the untagged sHsp versions (pBO1525, pBO1522, pBO1548 and pBO1521). Thereby, two amino acids, leucine and glutamic acid, were added at the carboxylterminal end of the sHsps. Plasmids for production of the hexahistidine-tagged versions were constructed by excision of the introduced stop codon via XhoI restriction and re-ligation (pBO1526, pBO1524, pBO1549 and pBO1523). Thus, beside the leucine and the glutamic acid, six histidines were added to the carboxyl-terminus of the sHsps. The P. putida ΔhspXYZ and ΔhspXYZΔibpA mutant strains were constructed as follows. An upstream fragment, ranging from −350 to +50 bp relative to the start codon of the hspX gene, and a downstream fragment, spanning from −50 to +350 bp according to the stop codon of hspZ, were amplified (primers listed in Supporting Information Table S2). Both fragments were ligated via the primer derived PstI sites and subcloned into EcoRI/HindIII sites of pUC18 (pBO1533). A gentamycin resistance cassette excised from plasmid pYPRUB5I was inserted into the PstI site between the upstream and downstream region (opposite orientation; pBO1538). The resulting fragment was transferred into suicide vector pEX18Tc via the primer derived EcoRI/HindIII restriction sites to obtain pBO1547. This plasmid was introduced into P. putida PG5 and P. putida ΔibpA by conjugation using the E. coli strain S17-1. Single cross-over integration mutants were selected on LB plates containing gentamycin and nalidixic acid. Single colonies were grown overnight in liquid LB without antibiotics and plated on LB containing gentamicin and 10% (w/v) sucrose to select for plasmid excision by double cross-over events. Gentamicin resistant and tetracycline sensitive colonies were checked for hspXYZ deletion by colony PCR and Northern analyses (data not shown).

RNA preparation Total RNA of cultured bacteria was isolated using the RNA preparation method described in Huntzinger and colleagues


Novel small heat shock proteins in Pseudomonas putida with minor modifications (Huntzinger et al., 2005). Five millilitre bacterial culture was mixed with 2.5 ml ice-cold stop buffer (100 mM Tris-HCl, pH 8, 200 mM β-mercaptoethanol, 5 mM EDTA) prior cell harvesting and washing with 1 ml ice-cold washing buffer (10 mM tris-HCl, pH 8, 100 mM NaCl, 1 mM EDTA). Following steps of RNA isolation were performed as described. Ribonucleic acid concentrations were determined with a NanoDrop spectrophotometer ND-1000 (peQlab, Erlangen, Germany).

Northern blot analyses Northern blot analyses were performed as described previously (Klinkert et al., 2012). Digoxigenin-labelled DNA probes were generated according to the instruction manual of the PCR DIG Probe Synthesis Kit (Roche, Basel, Switzerland) with primers listed in Supporting Information Table S2.

5′ RACE 5′ RACE was performed as described previously (Willkomm et al., 2005; Krajewski et al., 2013) with total RNA isolated from cells that were heat shocked for 10 min at 42°C. The gene-specific primer Pp_HspX_RACE_rv was used for reverse transcription, PCR amplification and sequencing. A control PCR was performed on genomic DNA and RNA used for 5′ RACE.

Enzymatic RNA structure probing RNAs for structure probing were synthesized in vitro by runoff transcription with T7 RNA polymerase, from EcoRVlinearized plasmids (Supporting Information Table S3). After purification and dephosphorylation, RNAs were labelled radioactively at the 5′ end (Waldminghaus et al., 2007). Partial digestions of labelled RNA with ribonucleases T1 (Ambion, Austin, USA) and nuclease S1 (Thermo Scientific, Waltham, USA) were conducted according to Waldminghaus et al., (2007), with the exception that supplied 5× reaction buffer for nuclease S1 and 5× TN buffer for RNase T1 (100 mM Tris acetate, pH 7.5, 500 mM NaCl) was used per reaction. Generation of alkaline ladder was conducted as described (Brantl and Wagner, 1994). Per reaction, 0.002 units RNase T1 (Ambion, Austin, USA) or 0.17 units nuclease S1 (Thermo Scientific, Waltham, USA) were applied. For orientation, a T1-ladder was set up by incubating 30 000 cpm labelled RNA in 1 μl sequencing buffer (Ambion, Austin, USA) at 90°C for 1 min followed by incubation with RNase T1 for 5 min at 45°C.

β-galactosidase activity assay Escherichia coli DH5α cells harbouring the bgaB plasmids (Supporting Information Table S3) were grown overnight in 10 ml LB at 25°C. 25 ml LB with ampicillin were pre-warmed to 25°C and inoculated with 2 ml overnight culture (OD600 ∼ 0.05). Cells were grown to an optical density (OD600) of 0.5 prior transcription induction with 0.01% L-arabinose (w/v; final concentration) and transfer of 10 ml of the culture to

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pre-warmed 100 ml flasks at 42°C. Thirty minutes after induction, 400 μl samples were taken and used for β-galactosidase assays as described (Gaubig et al., 2011).

Generation of HspY antisera Rabbit serum against His-tagged HspY was generated as follows. Escherichia coli BL21(DE3) cells harbouring the expression plasmid (pBO1549) were grown in LB containing kanamycin at 37°C. Protein synthesis was induced for 2 h at 37°C with 1 mM IPTG (final concentration). Bacterial cells were lysed by acceleration disruption (Constant cell disruptor TS; Constant System Limited, Daventry, UK) at 40 kpsi. C-terminally tagged HspY fusion protein was purified from cell lysate by Ni-NTA affinity chromatography according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Afterwards, HspY-His6 was further purified by size exclusion chromatography with a Superdex 200 10/300 GL gel filtration column (Amersham Biosciences, Freiburg, Germany). The purified protein was used for customized polyclonal antibody production (rabbit; BioScience, Göttingen, Germany).

Western analysis (immunodetection) According to their optical density (50 μl for OD600 = 1), cell pellets were re-suspended in protein sample buffer [final concentration of 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 20% glycerol, 50 mM Tris/HCl, pH 6.8]. Protein samples from affinity chromatography and size exclusion chromatography were mixed with protein sample buffer to achieve the final buffer concentration described above. Protein extracts were incubated at 95°C for 5 min, centrifuged (1 min, 13 000 rpm) and subjected to SDS gel electrophoresis (15% SDS polyacrylamide gels) and Western transfer using standard protocols. His-tagged proteins were detected using a Penta-His-HRP conjugate (Qiagen, Hilden, Germany). For immunodetection of HspY, polyclonal HspY antiserum was applied in a 1:10 000 dilution. Goat anti-rabbit immunoglobulin G(H+L)-HRP conjugate (Bio-Rad, Munich, Germany) used in a 1:3000 dilution served as secondary antibody. Chemiluminescence signals were visualized using enhanced chemiluminescence (ECL) Western blotting detection reagents (GE Healthcare, Munich, Germany) and ECL film exposure or chemiluminescence detector (FluorChem SP, Alpha Innotech, Biozym, HessischOldendorf, Germany).

Heterologous expression of sHsps in E. coli Escherichia coli BL21(DE3) cells harbouring the expression plasmids (Supporting Information Table S3) were grown in LB media containing kanamycin (50 μg ml−1) at 37°C. Once the cultures reached an OD600 of 0.5, production of the recombinant proteins was induced by addition of IPTG in a final concentration of 1 mM. 2–3 h after induction, bacterial cells were harvested and re-suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl) containing 0.25 mM protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride and DNaseI. Cells were disrupted by sonication (Branson signifier 250; Branson Ultrasonic,


1850 S. S. Krajewski, M. Joswig, M. Nagel and F. Narberhaus Danbury, USA) or by acceleration disruption (constant cell disruptor TS; Constant System Limited, Dave try, UK) at 40 kpsi. The lysate was centrifuged at 13 000 rpm, 4°C for 30 min to remove cell debris and filtered through a sterile filter (diameter 0.2 μm).

microlitre fractions were collected and analysed by SDSPAGE. The following standards were used to calibrate the column: thyroglobulin (669 kDa), amylase (200 kDa) and carbonic anhydrase (29 kDa).

Native PAGE Protein purification Purification of hexahistidine (His6)-tagged proteins was performed by affinity chromatography under native conditions with Ni-NTA resin (nickel-nitrilotriacetic acid; Qiagen, Hilden, Germany) pre-equilibrated with 50 mM NaH2PO4 and 300 mM NaCl. After application of the crude extract, the column was washed with washing buffer (50 mM NaH2PO4, 300 mM NaCl) containing increasing imidazole concentrations (20–75 mM imidazole). Purified proteins were eluted by raising the imidazole concentration up to 100–250 mM. Eluted proteins were analysed by SDS-PAGE on a 15% polyacrylamide gel. Protein concentrations were determined by Bradford assay (Bradford, 1976) using the Roti-Quant solution (Carl Roth, Karlsruhe, Germany) according to the manufacturer’s instructions with BSA as protein standard.

Co-purification of oligomers Oligomer formation of sHsps was analysed by co-purification assays based on the Ni-NTA affinity chromatography technique. Cells were lysed by sonication as described above. Before co-purification, aliquots of the crude extracts were subjected to SDS-PAGE to estimate the amount of synthesized sHsp and to use approximately equal amounts of each sHsp in the co-purification experiment. Crude extract containing a His6-tagged sHsp was mixed with the crude extract of an untagged sHsp and incubated for 2 h on ice to allow oligomer formation. Putative oligomers were purified via the His6-tagged protein in a small scale Ni-NTA purification. Therefore, 60 μl Ni-NTA resin were added to the sHsp mixture and incubated for 15 min on ice. Ni-NTA resin with the bound proteins were sedimented by centrifugation (2 min at 13 000 rpm). Ni-NTA bound proteins were washed four times with 500 μl washing buffer (50 mM NaH2PO4, 300 mM NaCl) containing increasing imidazole concentrations (20 mM, 30 mM, 50 mM and 75 mM). Purified proteins were eluted by addition of 70 μl buffer containing 250 mM imidazole. Elution fractions were analysed via SDS-PAGE. This procedure was performed with all possible combinations of His6-tagged and untagged proteins. As control, the same procedure was performed with tagged and untagged proteins alone.

Size exclusion chromatography (SEC) Native sizes of His6-tagged sHsp complexes were determined by analytical SEC. Purified proteins in elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole) were loaded on a Superdex 200 10/300 GL gel filtration column (Amersham Biosciences, Freiburg, Germany), preequilibrated with buffer (50 mM NaH2PO4, 300 mM NaCl). Separation was performed on ÄKTA explorer system (GE Healthcare) at 4°C at a flow rate of 0.3 ml min−1. Absorbance was recorded at a wavelength of 280 nm. Five hundred

Separation of native proteins and protein complexes was performed by native PAGE using 4–15% Mini-PROTEAN TGX Precast Gel (Bio-Rad) for use with Mini-PROTEAN electrophoresis cells according the manufacturer’s instruction. 7.5 μg of protein was mixed with the same volume of native PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% bromophenol blue), incubated for 25 min on ice, at RT or at 43°C, and then subjected to native gelelectrophoresis with native PAGE running buffer (25 mM TrisHCl pH 8.3, 192 mM glycine).

Thermal aggregation protection assay Thermally induced aggregation of the model substrate CS (Sigma-Aldrich, St Louis, USA) was measured in the presence or absence of various amounts of sHsps. Citrate synthase was dialysed against Tris/EDTA buffer (10 mM TrisHCl, 1 mM EDTA, pH 8.0). Thermal aggregation protection assay was performed in a reaction volume of 1 ml in 50 mM sodium phosphate, pH 6.8. CS (900 nM) was pre-incubated for 10 min at 43°C with varying concentrations of purified sHsp (225 nM, 450 nM, 900 nM and 1.8 μM) before start of the measurement. Citrate synthase aggregation was monitored as increased light scattering at 320 nm in an UV/Visspectrophotometer (Ultrospec 2100; GE Healthcare, Munich, Germany). Control measurements were performed with bovine α-crystalline (Enzo Life Sciences, Farmingdale, USA), BSA (Merck, Darmstadt, Germany) and purified sHsps in the absence of CS.

Bioinformatic methods Genome sequences were retrieved from the National Center for Biotechnology Information microbial genome database (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/ microbial_taxtree.html). Sequence alignments and phylogenetic tree were generated by the ClustalW2 software obtained from http:// www.ebi.ac.uk/Tools/msa/clustalw2/ (Larkin et al., 2007; Goujon et al., 2010). Phylogenetic tree was visualized with TreeDyn obtained from http://www.phylogeny.fr/version2_cgi/ index.cgi (Chevenet et al., 2006; Dereeper et al., 2008). BLASTp obtained from http://www.ncbi.nlm.nih.gov/ was used to search for homolog proteins and for calculation of amino acid identity and similarity (Altschul et al., 1990). Secondary structure predictions were performed with RNAfold (http://rna.tbi.univie.ac.at/) or RNAshapes (suboptimal folding; http://bibiserv.techfak.uni-bielefeld.de/ rnashapes/submission.html; Steffen et al., 2006; Bernhart et al., 2008; Gruber et al., 2008).

Acknowledgements We are grateful to Gerhard Burchhardt (Greifswald) for Pseudomonas putida PG5. We thank Ursula Aschke-


Novel small heat shock proteins in Pseudomonas putida Sonnenborn for excellent technical assistance, Daniel Momirovic for constructing plasmids pBO2962 and pBO2963 and Birgit Klinkert for construction of plasmids pBO2989, pBO2990 and pBO2992. Philip Möller, Birgit Klinkert, Johanna Roßmanith and Lisa-Marie Bittner are acknowledged for critical reading of the manuscript. This work was supported by grants from the German Research Foundation (DFG, SPP 1258: Sensory and regulatory RNAs in Prokaryotesand FN 240/10-1) to F.N.

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Supporting information Additional supporting information is available in the online version of the article at the publisher’s web-site. Fig. S1. Amino acid sequence comparison of the sHsps. Amino acids conserved in all sHsps are shown in dark grey. Conserved amino acids of the IbpA-like sHsps are shaded in light grey. The conserved I-X-I/V motif is marked by a black box. Fig. S2. Synthesis and solubility of HspX-His6. A. Synthesis of His6-tagged HspX. E. coli BL21(DE3) cells harbouring the expression plasmid were grown at 37°C to an OD 600 of 0.5. Samples for Western blot analysis were taken before and 2 h after addition of 0.25 mM IPTG. B. Solubility of HspX-His6. Crude extract containing HspX-His6 was centrifuged to separate soluble and insoluble protein. Pellet (P) and supernatant (S) fractions were analysed via Western blot analysis. Fig. S3. hspXYZ gene cluster in metabolically related bacteria. Genetic organization of the hspX, hspY and hspZ genes from Pseudomonas putida KT2440, Rubrivivax benzoatilyticus JA2, Geobacter sulfurreducens PCA, Acidovorax sp. CF316, Geobacter lovleyi SZ, Aromatoleum aromaticum EbN1, Methylococcus capsulatus str. Bath, Polymorphum gilvum SL003B-26A1, Methylobacterium extorquens AM1, Thioflavicoccus mobilis 8321, Anaerolinea thermophila UNI-1, Methylibium petroleiphilum PM1, Thioalkalivibrio sulfidophilus HL-EbGr7. Sequence identity and similarity of the deduced amino acid sequence to P. putida HspX, HspY and HspZ are given above the corresponding gene. Phylum affiliation is indicated by symbols. α: alpha-proteobacteria; β: beta-proteobacteria; γ: gamma-proteobacteria; δ: deltaproteobacteria; C: chloroflexi. Table S1. Strains used in this study. Table S2. Oligonucleotides used in this study. Table S3. Plasmids used in this study.


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