UDC 575 DOI: 10.2298/GENSR1501119M Original scientific paper

INACTIVATION OF Stigmatella aurantiaca csg A GENE IMPARES RIPPLING FORMATION

Ana MILOSEVIC-DJERIC 1, Susanne MÜLLER 2, and Hans URLICH SCHAIRER 3

1 Department of Gynaecology and Obstetrics, Hospital Centre Uzice, Uzice, Serbia 2 Univeristy of Iowa, Iowa City, USA 3Zentrum fur Molekulare Biologie, ZMBH, Heidelberg, Germany

Milosevic-Djeric A., S. Müller, and H. Urlich Schairer (2015): Inactivation of Stigmatella aurantiaca csgA gene impares rippling formation- Genetika, Vol 47, No. 1, 119-130. Stigmatella aurantiaca fruiting body development depends on cell-cell interactions. One type of the signaling molecule stigmolone isolated from S. aurantiaca cells acts to help cells to stay together in the aggregation phase. Another gene product involved in intercellular signaling in S. aurantiaca is the csg A homolog of Myxococcus xanthus . In close relative M. xanthus C signal the product of the csg A gene is required for rippling, aggregation and sporulation. Isolation of homologous gene in S. aurantiaca implicates a probable role of CsgA in intercellular communication. Inactivation of the gene by insertion mutagenesis caused alterations in S. aurantiaca fruiting. The motility behavior of the cells during development was changed as well as their ability to stay more closely together in the early stages of development. Inactivation of the csg A gene completely abolished rippling of the cells. This indicates the crucial role of the CsgA protein in regulating this rhythmic behavior. Key words: cell-cell signaling, development, fruiting body formation, myxobacteria, Stigmatella aurantiaca

INTRODUCTION Stigmatella aurantiaca belongs to the myxobacteria famous for their ability to form multicellular structures fruiting bodies in response to the starvation conditions ( SHIMKETS et al ., 2006; ZHANG et al., 2012; CLAESSEN et al ., 2014). Hundred thousand bacterial cells upon nutrient depletion migrate in concentric traveling waves, ripples, into aggregation centers and form three ______Corresponding author: Ana Milosevic-Djeric, Department of Gynecology and Obstetrics , Hospital Uzice, Milosa Obrenovica 17, 31 000, tel +38131 561255, Uzice, Serbia, mail: [email protected] .

120 GENETIKA, Vol. 47, No.1, 119-130, 2015 reassembling fruiting bodies consisting of a stalk bearing several sporangiols. Inside the sporangioles rod shaped vegetative cells differentiate into spherical resistant myxospores. Spores are generally more resistant than vegetative cells and can survive long starvation period ( SHIMKETS and BRUN , 2000). Some cells remain outside the fruiting bodies and after some time they undergo lysis that reflect programmed cell death ( NARIYA and INOUYE , 2008; BOYTON et al .., 2013). Formation of multicellular structures as well as differentiation of cells during development is regulated by extracellular signaling molecules ( KROOS , 2007; KAISER et al ., 2010). A fine coordination between cells depends on highly coordinated changes in gene expression during development ( KROOS and INOUYE , 2008). The most studied myxobacteria is Myxococcus xanthus which represents model organism to understand the molecular mechanisms underlying fruiting body formation ( HUNTLEY et al ., 2011). On the other hand Stigmatella is much more interesting than Myxococcus because it forms more complex fruiting bodies but it is also so much more demanding and much more harder to handle. This is the main reason for the small number of known information about this intriguing bacterium. Stigmolone is only one isolated chemical signal that acts in concentrations of about 1 nM to accelerate aggregation of Stigmatella cells at the beginning of development ( PLAGA et al ., 1998). Its activity depends on cell density and therefore it may have a role in “quorum sensing”. Additionally, cells have to be in direct contact or on the solid surface to secrete stigmolone. The stigmolone biosynthetic pathway as well as the putative pheromone receptor and signaling molecular mechanisms are still unknown ( PLAGA et al ., 1998). In contrast to S. aurantiaca in Myxococcus several signaling pathways were identified up to now. One of them C signal is necessary for rippling, aggregation, sporulation and expression of many developmental genes. All mutants unable to synthesized C signal carry mutation in the csg A gene. Transcription of the csgA gene is induced in response to starvation ( CRAWFORD and SHIMKETS , 2000). The csg A gene encodes 25 kDa protein that become proteoliticaly cleaved by the PopC protease to generate p17, the actual C-signal to 17 kDa protein ( LOBEDANZ and SØGAARD - ANDERSEN , 2003; SØGAARD -ANDERSEN 2008; ROLBETZKI et al ., 2008). PopC accumulates in vegetative cells and is secreted when cells are starving ( ROLBETZKI et al ., 2008). PopC secretion is prohibited by interaction with PopD and PopD is degraded in a RelA-dependent manner (KONOVALOVA et al ., 2012). According to the close phylogenetic relationship between S. aurantiaca and M. xanthus it was speculated that the CsgA protein might plays a role in communication between S. aurantiaca cells during development. To investigate this possibility an S. aurantiaca csg A insertion-mutant was constructed. Inactivation of the gene abolished rippling and caused alternation in cells migration pattern during aggregation.

MATERIALS AND METHODS Bacterial strains and plasmids The strains and plasmids used in this study are listed in Table .1. S. aurantiaca wild-type

DW4/3-1 cells were grown at 32°C in tryptone medium (1% Bacto Tryptone, 0.2% MgSO 4) supplemented with streptomycin sulfate (120 µg/ml) or with kanamycin sulfate (50 µg/ml) or oxytetracycline (7.5 µg/ml). Fruiting body formation and sporulation assays were done as previously described ( MÜLLER et al ., 2006). Escherichia coli strains were grown in Luria-Bertani or SOC medium at 37°C and supplemented with appropriate antibiotics as recommended by the manufacturer (Stratagene).

Electroporation of S. aurantiaca was performed as previously described ( STAMM et al ., 1999). A.DJERIC et al:Stigmatella aurantiaca csgA 121

Table 1. Bacterial strains and plasmids used in this study Strain or plasmid Description a Source or refe rence E. coli Stratagene XL1-Blue MRF´ (mcrA )183 ( mcrCB-hsdSMR- mrr )173 endA1 supE 44 thi-1 recA1 gyrA96 relA1 lac (F' proAB lacI q ZM15 Tn 10 ) (Tc r)

DH5 F´/ endA1 hsdR17 (rk- New England Biolabs mk+ )glnV44thi-1recA1gyrA (Nalr) relA1 (lacIZYAargF)U169deoR( 80 dlac (lacZ)M15)

S. aurantiaca DW4/3-1 Wild type (WT) (Sm r) AM8 WT; csgA ::pAM8 (Tc r Sm r) This study AM14 WT; neo ; 0,6k bp upstream of This study csgA (Km r Sm r) Plasmids pACYC177 Cloning vector (Km r) New England Biolabs pBR322 Cloning vector (Tc r) New England Biolabs pAM5 csgA in pACYC177 This study pAM8 csgA in pACYC177 This study pAM14 0,6 kbp upstream csgA :: trp- This study lacZ-neo in pBS SK – a Ap r, ampicillin resistance; Cm r, chloramphenicol resistance; Km r, kanamycin resistance; Sm r, streptomycin resistance; Tc r, tetracycline resistance; WT, wild type

DNA manipulations Standard genetic techniques for in vitro DNA manipulations and cloning were used. The method described by Neumann and co-workers was used for the isolation of total DNA from S. aurantiaca (MÜLLER et al ., 2006). Southern blot hybridization and detection were performed using the Biotin-Detection-System (Roche Diagnostics). Sequencing of the DNA was performed with ABI Prism tm 377 Sequence System (Perkin-Elmer Corporation) in the sequence facilities of the ZMBH.

RT-PCR The Access RT-PCR kit (Promega), which offers reverse transcription and PCR amplification in a single reaction, was used to detect mRNA expression. First strand cDNA synthesis was performed by incubation at 48°C for 45 min, thereafter the reaction was incubated at 94°C for 2 min to inactivate AMV reverse transcriptase and to denature the RNA/cDNA/primer mixture. Second strand cDNA synthesis and the amplification followed directly throught 40 122 GENETIKA, Vol. 47, No.1, 119-130, 2015 thermal cycle (30 sec at 94°C, 1 min at 54°C and 1 min at 68°C). This was followed by a final extension for 7 min at 68°C. After electrophoresis on an agarose gel, the cDNA was stained with the ethidiumbromide for 30 min, and analysed by UV illumination. Pimer pair pair used was csgA7CTGGATGTGCTCATCAAC/csgA8CTGGAAGTCGAGGAACATG.

Construction of plasmid pAM8 and mutant AM8 Plasmid pAM8 was generated by digesting pAM5, plasmid that contains the csg A locus, (MILOSEVIC , 2003), with the Sac I and Sph I in order to remove the internal part (340 bp) of the csg A gene. After restriction 5’-overhanging ends were filled using T4 DNA polymerase and ligate to tetracycline resistance gene amplified by PCR from pBR322 with the primer pair Tcfw Xba and Tcrv Xba . Plasmid pAM8 was integrated into the genome of S. aurantiaca wild type by electroporation. Oxytetracycline resistance clones were selected and double recombination event was confirmed by Southern analysis.

Construction of plasmid pAM14 and mutant AM14 Plasmid pAM14 was constructed by ligation of 0,9 kbp fragment harbouring 0,6 kbp of the csg A promoter, upstream and 0,3 kbp downstream of putative ATG start codon amplified by PCR (primer pair Not I csgA 21 and csgA 20 Xba I), to plasmid pSM62 (kindly provided by S. Müller). Plasmid pSM62 (derivative of pBSSK-) contains the promoterless trp Alac Z gene fused to a neo cassette for selection of recombinants after transformation. The plasmid pAM14 sequence was reconfirmed by restrictional analysis and sequencing. In order to produce a csg A merodiploid mutant strain, a plasmid pAM14 was integrated into the S. aurantiaca wild type csg A locus by a single recombination event. Kanamycin-resistant clones were selected and single recombination event was confirmed by Southern blot (data not shown) resulting in AM14. ß-Galactosidase activity in strain AM14 was determined under vegetative and developmental conditions as described previously. Fifty micrograms of total protein in 0.1 ml MOPS (morpholinepropanesulfonic acid) buffer (50 mM MOPS, pH 7.5, 10 mM MgCl 2, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) was mixed with 0.3 ml Na phosphate buffer (10 mM Na phosphate, pH 7, 0.1 M NaCl methylumbelliferyl, 1 mM MgCl 2) containing the fluorescent substrate 4-MUG (4- ß-D-galactopyramoside). After incubation at 37°C for 30 min the reaction was stopped with 3 ml of 0.1 M glycine buffer (pH 10.3). The substrate was cleaved by ß- galactosidase releasing the fluorescent 4- methylumbelliferon (4-MU) that was measured at an excitation wavelength of 360 nm and an emission wavelength of 450 nm using a Shimadzu RF 5000 fluorescence spectrophotometer.

RESULTS Inactivation of S. aurantiaca csgA gene and physiological function of CsgA The csg A knock out strain was constructed (AM8) by insertion mutagenesis. Plasmid pAM8 harboring disrupted csg A allele with the tetracycline resistance cassette in divergent transcriptional orientation was integrated into the genome by double recombination event (Fig.1a). To investigate possible effects of csg A inactivation AM8 mutant cells were assayed for the ability to form fruiting bodies. The phenotype of the mature mutant fruiting body was the same as the wild type (Fig.1b). Furthermore, AM8 cells retained ability for sporulation. Difference was observed in localization of aggregating centers and mature fruiting bodies during fruiting body formation assay. In contrast to wild type mutant cells migrate more inside of spots forming an A.DJERIC et al:Stigmatella aurantiaca csgA 123 aggregation centers in outside ring (Fig.2). With progression of the development from 8 to 12 h the first differences between the two strains could be observed. During the indicated time period mutant cells migrated to the outer part of the spot forming a circle with high cell density. Unlike mutant cells, wild type cells concentrated mostly in the inner part of the spot and the edge of the spot was transparent as at the beginning (Fig.2). From 12 to 20 h mutant cells continued to accumulate in an outer circle. They formed aggregation centers very close to each other in the outer ring. Importantly specific rippling trails were not observed with the mutant cells during this time period. Rippling is highly organized cells movements in concentric traveling waves that precede aggregation. Wild type cells formed many aggregation centers between 12 and 20 h from which fruiting bodies will arise in later stages. Aggregation of the wild type cells was also visible in the inner part of the spot. This is not the case during aggregation of the mutant cells. The rippling was well visible when analyzing development of wild type cells. After 20 to 26 h mutant as well as wild type cells formed fruiting bodies. No changes in the appearance of the fruiting bodies were observed after 48 h. The mutant fruiting bodies were located in the outer ring of the circle whereas the wild type ones were also present in the inner part of the circle. Inactivation of the csg A gene resulted in alternation in cell migration as mutant cells completely lost ability for rippling but they were still able to develop wild type fruits.

Fig.1a Construction of the csgA insertion mutant. Fig.1b Side-view of the representative fruiting body formed by the csgA insertional mutant (AM8) and the wild type

124 GENETIKA, Vol. 47, No.1, 119-130, 2015

Fig.2 Developmental phenotype of csgA insertional mutant versus wild type. Cells were exposed to starvation on water agar plates for indicated period of time. Spots were viewed from above. A.DJERIC et al:Stigmatella aurantiaca csgA 125

Fig. 3. Expression of csgA detected by RT-PCR. The RT-PCR reactions were carried out using the same amounts of total RNA. Lane 1: 100 bp DNA Ladder (New England BioLabs); lane 2: control provided by kit (Promega); lane 3: vegetative cells; lane 4: indol induced sporulation after 10 min; lane 5: indol induced sporulation 30 min; lane 6: indol induced sporulation 60 min; lane 7: indol induced sporulation 120 min, lane 8: cells after 8h of development; lane 9: cells after 20h of development, lane 10: cells after 30h of development.

Expression of csgA gene in S. aurantiaca Transcription of the csgA gene in S. aurantiaca during vegetative growth, development, or artificially induced sporulation was determined by qualitative RT-PCR. Total RNA isolated from cells under different conditions was transcribed with AMV reverse transcriptase and cDNA was amplified with primer pair csgA7/csgA8 as described in the method part. The transcription of csg A was detected during vegetative growth as well as during development (Fig.3). The csg A expression was detected after 8, 20, 30 h of development. Additionally expression was also detected during artificially induced sporulation with indol after 10, 30, 60 and 120 min. To study the level of the csg A expression a promoter region of csg A was fused to trp A- lac Z reporter gene. Plasmid pAM14 containing promotor csg A- trp A- lac Z-neo fusion was integrated into the genome by a single recombination event leading to a merodiploid mutant AM14 with a wild type csg A allele and a csg A promotor- trp A- lac Z-neo fusion allele in tandem (Fig.4 a). Mutant AM14 displayed the same behavior during development and formed wild type fruiting bodies enclosing viable spores (Fig.4 b). This led to the conclusion that the upstream region of 0,6 kbp used to construct the merodiploid mutant is sufficient for expression of the csg A gene. -Galactosidase activity was determined in AM14 cells during vegetative growth and development as describes in material and methods. The merodiploid mutant cells showed a very low level of -galactosidase activity during vegetative growth with a slight increased during progression of starvation (about 8h) (Fig.4 c). Additionally, -galactosidase was determined in situ by placing AM14 cells on starvation agar supplemented with X-gal. Starvation of the AM14 cells resulted in fruiting bodies whose stems were stained in blue (Fig.4 d). Taking together these results indicate very low expressional level of csg A gene under vegetative and developmental conditions 126 GENETIKA, Vol. 47, No.1, 119-130, 2015

Fig. 4a Construction of the merodiploid strain AM1. Fig. 4b Side-view of representative fruiting bodies formed by AM14 and wild type after 48h of starvation on water agar. Fig. 4c Determination of the b-galactosidase activity of strain AM14 during fruiting body formation (50 mg). Fig. 4d Fruiting body of the merodiploid strain AM14 on starvation agar containing X-gal.

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DISCUSSION Differential gene expression in the time course of development is tightly regulated by a sophisticated communication network in S. aurantiaca. In contrast to the other well-studied myxobacterium M. xanthus research on intercellular signalling in S. aurantiaca is just at the beginning . Stigmolone, a pheromone, is the only isolated and characterized signal molecule of S. aurantiaca . In addition, several genes were identified whose products might have a role in cell-cell communication ( MÜLLER et al ., 2006). One of the previously identified genes, homolog to the M. xanthus csg A, was further characterized in this work. The C signalling pathway is the most A.DJERIC et al:Stigmatella aurantiaca csgA 127 intensively studied in M. xanthus . C signal is necessary for regulation of the cells movement behavior that results in the formation of rippling waves and aggregation of the cells. Whereas rippling is completely abolished in the csg A mutant, aggregation is impaired under certain conditions. Unlike wild type cells, which aggregate into compact translucent mounds after 12 h of starvation on the agar surface, csg A mutants aggregate only after 18 h into larger, less compact mounds and ridges. The C signal acts about 6 h after the beginning of development. A low concentration of the C signal is required for rippling and aggregation. A higher concentration induces sporulation and C signal dependent gene expression including csg A itself. Therefore the model of the C signalling pathway indicates two branches. One branch leads to the regulation of the movement responds of cells and the other branch controls sporulation and expression of the late developmental genes. The function of the S. aurantiaca CsgA protein in intercellular communication was elucidated by inactivation of the gene. The detectable phenotype that was observed as a consequence of csg A inactivation by insertion mutagenesis suggests an involvement of CsgA in intercellular communication of S. aurantiaca. Inactivation of csg A in S. aurantiaca impaired rippling, mutant cells showed somehow altered migration and aggregation patterns during development, whereas the shape of the fruiting body showed no obvious differences as compared to the wild type. Additionally, mutant cells differentiated into viable myxospores enclosed in the fruiting bodies. The expression of the csgA gene in S. aurantiaca was determined by qualitative RT-PCR. The csgA mRNA was detected in vegetative cells and also during development. Analysis with promoterless trp A-lac Z gene as reporter gene indicated that the csg A was expressed at a low level vegetatively with slight increase during development in mutant AM14. Also determination of the in situ beta galactosidase activity as well as RT PCR indicate that the csg A gene is expressed during development in S. aurantiaca. This result shows that CsgA protein is required for formation of rippling wave pattern during S. aurantiaca development. Rippling is observed only in myxobacteria and it is organized cells movement in concentric equidistant ridges ( BERLEMAN and KIRBY , 2009; ZHANG et al ., 2011). Myxococcus xanthus cells self-organize into periodic bands of traveling waves, termed ripples, during multicellular fruiting body development and predation on other bacteria ( ZHANG et al ., 2012b). In M. xanthus rippling dependents on CsgA ( SAGER and KAISER , 1994) . C-signal acts on top of a signaling pathway regulating developmental gene expression and motility (GRONEWOLD and KAISER , 2007). The C-signal suppresses cell reversals in a cell-cell contact dependent manner. As a result cells stream into aggregates forming fruiting bodies that contain dormant spores. The cell surface C-factor activates a receptor on neighboring cells causing the suppression of cell reversals through methylation of FrzCD, a receptor of the Frz chemosensory pathway ( ZHANG et al ., 2012a). Another consequence of the csg A inactivation in S. aurantiaca was that unlike wild type cells that stay more together in the early stage of development, mutant cells were unable to stay together. Thus, a signal or signals produced by the CsgA protein might contribute in sensing the density of the cells prior to the aggregation stage. Additionally, the csg A mutant cells did not respond to stigmolone in a bioassay performed on agar. This indicates possible connection between CsgA and stigmolon. The increase of the aggregation rate was not observed with csg A mutant cells in the stigmolone bioassay in contrast to acceleration of the aggregation rate in an 128 GENETIKA, Vol. 47, No.1, 119-130, 2015 assay performed with the wild type cells. Therefore one possibility might be that stigmolone is a substrate for CsgA in S aurantiaca. In that case the reduction of the keto group in stigmolone by CsgA might lead to a real signal molecule, stigmolol, which is exchanged between the cells during development. To investigate this possibility it would be necessary to purify the putative stigmolol from wild type cells. The csg A mutant cells would not have stigmolol but the mutant phenotype could be rescued by adding this substance. M. xanthus does not respond to stigmolone by accelerating fruiting body formation in a bioassay ( PLAGA et al ., 1998). As mention before in S. aurantiaca forms complex fruiting bodies that consist of a branched stalk bearing several sporangioles, whereas M. xanthus forms only mounds filled with spores. Thus, complexity of S. aurantiaca fruiting bodies implicates requirements for a more subtile communication network in S. aurantiaca . It is also possible that there is a way to bypass the csg A mutational block in S. aurantiaca since the pressure to survive under nutrient limitation is high. It might be that mutant cells adopt some suppressor mutations that allow them to bypass the csg A block. It is known that overproduction of SocE in M. xanthus csg A mutant cells bypasses the csg A mutation and cells are able to aggregate and to produce fruiting bodies in the absence of C signaling ( CRAWFORD and SHIMKETS , 2000). However, this study revealed connection between cell motility behavior and csg A gene and open possibility for further investigation of signaling molecular mechanism in S. aurantiaca . To fully understand the CsgA dependent modulation of cell behavior it will be also necessary to analyse the effect of the C-signal in the context of other cell-cell interactions in S. aurantiaca during the complex developmental process.

ACKNOWLEDGMENTS Thanks Richard Herrmann, Wulf Plaga, Diana Hofman for support and helpful discussions. This research was supported by the Deutsche Forschungsgemeinschaft. Received September15 th , 2014 Accepted January 28 th , 2015

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INAKTIVACIJA Stigmatella aurantiaca csg A GENA ONEMOGU ĆAVA RITMI ČKO KRETANJE

Ana MILOŠEVI ERI 1, Susanne MÜLLER 2 i Hans Urlich SCHAIRER 3

1Ginekologija i akušerstvo, Zdravstveni Centar Užice, Užice, Srbija 2Universitet Iowa, Iowa, SAD 3Centar za molekularnu biologiju, ZMBH, Heidelberg, Nema ka

Izvod Stigmatella aurantiaca je myxobacteria poznata po formiranju više eliskih struktura kao odgovor na nepovoljne uslove životne sredine. U okviru višeeliskih formacija koje podse aju na strukturu drveta vegetativne elije se diferenciraju u višestruko otporne spore koje preživljavaju negativne uslove spoljašnje sredine. S obzirom na neobi an životni ciklus ove bakterije, ona predstavlja dobar model sistem za prou avanje gena i proteina uklju enih u me uelijsku komunikaciju. Druga myxobacteria koja je poznatija i bolje prou ena je Myxococcus xanthus. Kod M. xanthus-a product csg A gena je neophodan za me uelijsku komunikaciju. U ovom radu je prikazana uloga csgA gena kao i CsgA proteina u interakciji me u elijama Sigmatelle u toku razvoja više eliskih struktura i formiranja visoko rezistentnih myxospora. Primljeno 15. IX. 2014. Odobreno 28. I. 2015.