Bacterial strains and plasmids used are listed in Table 1. HP15 was isolated from particles collected from surface waters of the German Bight (Grossart et al., 2004). The bacterium was grown in marine broth (MB) medium (ZoBell, 1941) using sterile North-Sea water at 28°C. MB medium was supplemented with uracil (5 μg ml⁻¹) as a pyrimidine source for growth of HP15 pyrimidine-deficient auxotrophs. During the in vitro screening, MB medium was supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (50 μg ml⁻¹) allowing for blue/white colony selection. f/2 GLUT medium was made with f/2 medium (Guillard and Ryther, 1962) supplemented with 5 g l⁻¹ glutamate and was used as minimal medium to confirm auxotrophy of the HP15 mutant ΔpyrB. f/2 medium was prepared with pre-filtered (0.2 μm pore size) and autoclaved North-Sea water. All herein reported CFU/ml values for bacteria were derived by determining the optical density at 600 nm (OD₆₀₀) and deducing CFU/ml from a previously established calibration curve of OD₆₀₀ vs. CFU/ml derived from serial dilution platings.

Escherichia coli strains were maintained in Luria-Bertani (LB) agar medium supplemented with appropriate antibiotics. E. coli DH5α was used for maintaining the HP15 genomic library generated in the IVET vector. E. coli ST18 (Thoma and Schobert, 2009) was used as a donor strain during bi-parental conjugation and grown in LB medium containing 50 μg/ml 5-aminolevulinic acid (ALA). E. coli HB101 (pRK2013) was used as a helper strain during tri-parental conjugation (Figurski and Helinski, 1979). The following antibiotics were added to media when needed (in μg ml⁻¹): chloramphenicol, 25; kanamycin, 50; and ampicillin, 50.

Axenic cultures of T. weissflogii (CCMP 1336) were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (Maine, USA). Diatom cultures were grown at 16°C in f/2 medium, with a 12-h photoperiod at 115 μmol photons m⁻² s⁻¹ and were checked regularly for bacterial contaminations by plating on LB agar medium plates and by frequent microscopic observations. Diatom cell numbers were determined by cell counts in a Sedgewick Rafter Counting Chamber S50 (SPI Supplies, West Chester, PA, USA).

Plasmid preparation, DNA extraction, PCR, and other standard DNA techniques were performed as previously described (Sambrook et al., 1989). Restriction enzymes and DNA-modifying enzymes were used as recommended by the manufacturer (Fermentas, St. Leon-Rot, Germany). Nucleotide sequencing was carried out at Eurofins MWG (Ebersberg, Germany). DNA sequences analysis and oligonucleotide primer design were done using Vector NTI^®; Software 10.3.0 (Invitrogen Corporation, Carlsbad, CA, USA). Nucleotide and amino acid sequences were compared using the Basic Local Alignment Search Tool BLAST provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1990) and InterProScan Sequence Search provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/pfa/iprscan/). The oligonucleotides used in this study are listed in Table 2.

The first step to implement the IVET strategy is the construction of an IVET vector (Supplementary Figure 1); the remaining steps in the strategy are summarized in Figure 1. The broad-host-range plasmid pBBR1MCS-4 (Kovach et al., 1995) was used as a backbone to construct the IVET vector (Supplementary Figure 1). The full-sized E. coli promoter-less lacZ gene was amplified from plasmid pMC1871 with the primers LacZF/LacZR (Smirnova and Ullrich, 2004). The resulting 3.1-kb PCR fragment was treated with KpnI and ligated into KpnI-treated pBBR1MCS-4 in opposite direction of the vector-borne lac promoter resulting in plasmid pITM3. A second plasmid, pITM4, was generated with the 3.1-kb PCR fragment containing lacZ cloned in the same orientation as that of the lac promoter of pBBR1MCS-4. Plasmids pITM3 and pITM4 were transformed into E. coli ST18 and subsequently transferred to HP15 by conjugation. For this, cells of donor and recipient (~10⁹ cell ml⁻¹ each) were mixed in a ratio of 1:2, spotted on LB agar plates, and incubated for 24 h at 28°C. The cell mass was then scraped off and re-suspended in MB medium for subsequent dilution plating. Transconjugants were selected on MB agar supplemented with ampicillin and X-Gal after incubation at 28°C for 2–3 days. Presence of IVET plasmids in transconjugants was confirmed by plasmid DNA isolation using the Midi Prep Kit according to the manufacturer's recommendations (Macherey-Nagel, Düren, Germany) and agarose gel electrophoresis.

Primers pyrBF and pyrBR were designed to amplify the promoter-less pyrBC locus from HP15. This 2,285-bp PCR product was restricted with HindIII and XhoI and cloned into HindIII/XhoI-treated pITM3 to generate pITM3_pyrB. Plasmid pITM3_pyrB was transformed into HP15 wild-type and ΔpyrB mutant by bi-parental conjugation as described above. The transconjugants were selected in MB media containing X-Gal and ampicillin.

A gene-specific mutagenesis based on homologous recombination was conducted to generate a ΔpyrB mutant in HP15 according to Hoang et al. (1998) (Figure 1A). A mutagenic plasmid was constructed in which a chloramphenicol resistance (Cm^R) cassette was flanked by DNA fragments obtained from upstream and downstream regions of the pyrB gene as follows: 960 bp upstream and 984 bp downstream of the pyrB gene were PCR-amplified using the primer pairs pyrB_upF/pyrB_upR and pyrB_down2F/pyrB_down2R, respectively. Both flanking fragments were sub-cloned into pGEM®-T Easy vector (Promega, Mannheim, Germany) resulting in plasmids, pGEM_pyrBup and pGEM_pyrBdown, respectively. A DNA fragment of 1,129 bp carrying a Cm^R cassette was amplified with the primers cm2R/cm2F from pFCM1. The fragment was treated with NdeI and sub-cloned into NdeI-treated pGEM_pyrBdown resulting in plasmid pGEM_pyrB_down_cm. From this plasmid, the 2,168-bp fragment containing the downstream region and the Cm^R cassette was restricted with KpnI/NheI and ligated into KpnI/NheI-treated pGEM_pyrB_up, resulting in plasmid pGEM_pyrB_down_cm_up. From this plasmid, a 3,134-bp fragment containing both flanking regions and the Cm^R cassette was excised with HindIII and ligated into the HindIII-treated vector pEX18Tc. The generated conjugable mutagenic construct pEX_pyrBcdu was then transformed into HP15 by bi-parental conjugation. Resulting mutants were selected on chloramphenicol-containing MB agar plates. A successful double cross-over event for the ΔpyrB mutant was confirmed by PCR using the primers mut_pyrBF/mut_pyrBR resulting in the expected 1,205 and 1,410-bp amplification fragments for the wild-type and the mutated pyrB gene, respectively.

To confirm the auxothrophy of mutant ΔpyrB, it was grown in f/2 GLUT medium with and without uracil. Subsequently, to determine whether the pyrB gene is a suitable selection marker for promoters expressed during the interaction with T. weissflogii, mutant ΔpyrB and the wild-type were separately co-cultivated with the diatom in the absence of uracil. Bacteria were grown overnight in MB liquid medium at 18°C and the cells were harvested by centrifugation at 4,000 rpm for 15 min. To avoid carrying over of nutrients from MB medium the cells were washed twice with f/2 medium. Cell numbers were adjusted to ~10⁵ CFU ml⁻¹ and mixed with ~10⁴ cells ml⁻¹ of T. weissflogii in exponential growth stage. The cultures were incubated for 6 days at 16°C, with a 2-h photoperiod at 115 μmol photons m⁻² s⁻¹, and shaking at 50 rpm. Dilutions series were done daily in appropriate media, and CFU numbers were determined.

To complement the pyrB mutant, a functional native promoter from the HP15 genome was cloned upstream the pyrBC-lacZ fusion in pITM3_pyrB. Such a native promoter was selected by partial restriction of the genomic DNA of HP15 with Sau3AI and cloning of a pool of restriction fragments into BamHI-treated plasmid pITM1 (Sonnenschein et al., 2011). The resulting genomic library was transformed into HP15 wild-type by bi-parental conjugation. Five blue colonies were selected and the region upstream the pyrBC-lacZ fusion from one of those clones (pITM1_Sau3A_3) was sequenced thereby identifying the native promoter. This DNA region was excised from pITM1_Sau3A_3 with XbaI and HindIII and cloned into XbaI/HindIII-treated pITM3_pyrB, resulting in the plasmid pITM3_pyrB_prom, which was transformed into ΔpyrB HP15 by bi-parental conjugation. Transconjugants were selected after incubation on f/2 GLUT agar supplemented with ampicillin and X-Gal without uracil at 28°C for 2–3 days.

A genomic IVET library of HP15 was constructed in pITM3_pyrB as follows (Figure 1B): Total genomic DNA of HP15 was partially digested with Sau3AI; DNA fragments with sizes ranging from 0.5 to 1.5 kb were selected, and ligated into the BamHI site of pITM3_pyrB. The IVET library was then transformed into E. coli DH5α. To verify the randomness of inserts, 24 clones of E. coli DH5α were randomly picked and the region upstream the pyrBC-lacZ fusion was amplified with the primers T7 and PyrB_out by colony PCR. The IVET library was then transferred into the mutant ΔpyrB by tri-parental conjugation. Briefly, E. coli DH5α carrying the IVET library was used as a donor and E. coli HB101 (pRK2013) served as a helper strain. Cells of donor, helper, and recipient (~10¹⁰ cells ml⁻¹) were mixed in a ratio of 1:1:2, spotted on LB ALA agar plates, and incubated for 24 h at 28°C. Cell mass was scraped off and re-suspended in MB medium for subsequent dilution plating. The resulting transformants were selected on MB agar plates containing chloramphenicol, ampicillin, uracil, and X-Gal. To verify the randomness of plasmids transformed into mutant ΔpyrB, 24 clones were randomly picked and subjected to colony PCR as described above. Although, cloning by partial digestion using an enzyme with frequent recognition sites does not comprehensively cover the entire genome of M. adhaerens, the randomness of insert sizes allows to conclude that a genome-wide array of promoter sequences had been obtained.

For the in co-culture selection of promoters, co-cultivation of mutant ΔpyrB transconjugants carrying the IVET library with diatom cells was conducted (Figure 1C). Briefly, 10,000 cells ml⁻¹ of T. weissflogii in exponential growth stage were mixed with the bacterial transconjugants (1 × 10⁵ CFU ml⁻¹) in cell culture flasks with a final volume of 35 ml of f/2 media and incubated at room temperature and a 2-h photoperiod for 24 h. Next, 5 ml of the co-cultures were transferred to 30 ml fresh f/2 medium containing T. weissflogii in exponential growth stage (10,000 cells ml⁻¹) and incubated under the same conditions as above for additional 24 h. This procedure was repeated five times in order to enrich for positive clones. After 6 days of incubation, transconjugants were recovered and subjected to in vitro selection. For this, cultures initiated on day 4 and 5 were individually centrifuged at 4,000 rpm for 20 min and dilution series were performed with recovered cells in MB agar plates containing chloramphenicol, ampicillin, uracil and X-Gal at 28°C. After 3–4 days of incubation brownish colonies (non-blue) were selected and re-streaked.

A control experiment was performed to observe the growth pattern of the mutant ΔpyrB carrying plasmid pITM3_pyrB without any insertion upstream the pyrBC-lacZ fusion (empty IVET vector). 10,000 cells ml⁻¹ of T. weissflogii in exponential growth stage were mixed with ΔpyrB transconjugants carrying pITM3_pyrB (1 × 10⁵ CFU ml⁻¹) in cell culture flasks in f/2 medium and incubated at room temperature and a 2-h photoperiod for 5 days, which was used in all previous studies on this model interaction system (Gärdes et al., 2011) because the diatom aggregate formation was optimal under this light regime. Dilution series were carried out daily from the culture and the number of CFU was calculated.

Brownish colonies of ΔpyrB transconjugants were selected after co-inoculation with T. weissflogii, and subjected to colony PCR with the primers T7/pyrB_out to check for the presence of an insert upstream of the pyrBC-lacZ fusion in pITM3_pyrB. Transconjugants giving individually different PCR products of sizes larger than 570 bp were selected and their PCR product sequences determined. The obtained sequences were analyzed by BLAST and potential promoters regions as well as downstream located genes identified. The positive transconjugants were individually co-inoculated with T. weissflogii to confirm their survival in presence of diatoms. For this, bacteria were grown overnight in MB liquid cultures at 18°C, the cells were harvested by centrifugation at 4,000 rpm for 15 min, washed twice with f/2 medium, their concentration adjusted to 1 × 10⁵ CFU ml⁻¹, and mixed with 10,000 cells ml⁻¹ of T. weissflogii in exponential growth stage. The co-cultures were incubated for 8 days at 18°C, with a 2 h photoperiod. Dilutions series were done in appropriate media and CFU numbers were determined.

For the identification of bacterial promoters, the gene pyrB was used as selection marker. pyrB is essential for survival encoding for aspartate transcarbamoylase (ATCase; EC 2.1.3.2), an enzyme that is necessary for biosynthesis of pyrimidines (Schurr et al., 1995). The availability of pyrimidines in many natural environments is limited, which renders pyrB a successful selection marker (Lee and Cooksey, 2000). To verify its potential as a selection marker in our system, a pyrB mutant in HP15 was created. This mutant should be unable to grow in the absence of a pyrimidine source. A mutagenic construct was transformed into HP15 wild-type and the pyrB gene was replaced by a Cm^R cassette via homologous recombination. A clone that had undergone a double crossover of the Cm^R cassette as demonstrated by PCR was selected. Subsequently, the auxotrophy of the pyrB deletion mutant was confirmed by testing its growth on agar minimal medium with or without uracil as the sole pyrimidine source. Mutant ΔpyrB was not able to grow without uracil thus proving its expected phenotype (data not shown). In addition, growth of mutant ΔpyrB and HP15 wild-type in liquid f/2 GLUT medium were indistinguishable from each other when uracil was supplemented indicating that the mutation did not affect bacterial growth in general (Supplementary Figure 2). However and as expected, mutant ΔpyrB did not grow without uracil.

To determine whether lack of pyrB expression is a suitable selection criterion during the interaction with T. weissflogii and to test whether the diatom provides any pyrimidines to functionally complement the mutant's genotype, mutant ΔpyrB was cultivated with diatoms in f/2 medium with and without any uracil. Mutant ΔpyrB cell numbers dramatically decrease during the incubation time when compared to the growth of the wild-type when no uracil was added to the medium (Figure 2). In accordance with the in vitro growth, mutant ΔpyrB grew similar to the wild type in presence of diatoms and uracil. These result clearly demonstrated that the diatom is not providing any pyrimidines proving pyrB's suitability as a selection marker gene for this study.

The suitability of lacZ as an in vitro reporter gene in HP15 had previously been described (Sonnenschein et al., 2011), thus this gene was used for the construction of the IVET vector. As an in co-culture selection marker the promoter-less pyrBC locus of HP15 was chosen since it had been previously shown that the gene pyrB alone was not able to complement pyrB deletion mutants and that the pyrC gene located downstream of pyrB was required for this purpose (Schurr et al., 1995; Lee and Cooksey, 2000). In order to be suitable tools, the ΔpyrB mutant must not grow unless provided by uracil while the IVET plasmid must not complement the ΔpyrB mutant as long as it does not carry a functional promoter sequence upstream of the in co-culture selection marker gene. The ΔpyrB mutant carrying the IVET plasmid pITM3_pyrB did not grow in f/2 GLUT medium without uracil. In contrast, this transconjugant grew well in form of brownish colonies in f/2 GLUT medium supplemented with uracil and X-Gal (data not shown) confirming that the pyrBC-lacZ fusion present in pITM3_pyrB is not expressed. Furthermore, the introduction of a functional promoter derived from the genome of HP15 into the IVET plasmid and its subsequent transformation into ΔpyrB mutant allowed its full complementation. Corresponding transformants grew on agar medium without uracil and formed LacZ-expressing (blue) colonies when X-Gal was provided (data not shown). In summary, these tested parameters demonstrated that the IVET plasmid and the ΔpyrB mutant were suitable tools for the identification of diatom contact-induced genes of HP15.

A genomic library of HP15 was constructed in pITM3_pyrB and transformed into E. coli DH5α plated on 50 agar plates each containing 200–300 colonies resulting in a total of roughly 10,000 to 15,000 clones. The randomness of inserts was confirmed by PCR with 190 randomyl chosen clones showing that 85% of the analyzed E. coli DH5α transformants had an insert in the IVET plasmid. Importantly, all of the insert sizes were different from each other (data not shown) demonstrating that the IVET library contained a random number of different DNA fragments. After conjugation of the IVET library into mutant ΔpyrB, a total of 4.5 × 10⁶ CFU ml⁻¹ were obtained, with roughly 40% of the transconjugants forming LacZ-positive blue colonies on X-Gal agar medium. To demonstrate the randomness of IVET plasmids in the transconjugant pool, a second round of PCR tests was done. In this case 82% of the analyzed 200 clones had an insert, and all inserts had different sizes (data not shown) suggesting that roughly one fifth of all transferred IVET plasmids did not contain an insert. In conclusion, there results hint at a randomness of DNA fragments inserted in the IVET plasmid but do not allow speculations about the completeness of the screen since the very frequently occuring recognition sites of the used endonuclease Sau3A might have been used unevenly during the applied partial digest.

In order to test whether or not mutant transconjugants with empty IVET plasmids could survive the in co-culture selection, first a control experiment was conducted, in which a ΔpyrB transconjugant carrying an empty IVET plasmid was grown together with the diatom. From an initial inoculum of 1.2 × 10⁵ CFU ml⁻¹ only 395 CFU ml⁻¹ were recovered after 2 days and 22 CFU ml⁻¹ after 3 days. However, after day 4 no colonies were observed. These results showed that transconjugant ΔpyrB (pITM3_pyrB) is not able to survive for more than 4 days in the presence of the diatom. Consequently, the following diatom-bacteria co-cultures were performed in such a way that positive clones were enriched while negative clones or clones with empty IVET plasmids were eliminated. To frequently refresh potential diatom-borne selecting factors or signals, co-cultures of ΔpyrB mutants carrying the IVET library with diatom cells were diluted every 24 h by transferring 5 ml of the co-culture into f/2 medium containing fresh diatom cells. Co-cultures were repeated several times to increase the number of positive clones. Determination of CFUs was carried out daily (data not shown), and randomly chosen brownish colonies (non-blue) were selected while blue colonies, indicating a constitutive expression of lacZ, were discarded. Brownish colonies represented transconjugants with IVET plasmids carrying a promoter sequence inactive under in vitro conditions. However, since these transconjugants had survived several diatom co-cultures, their promoter sequences must have been active in co-culture. All obtained brownish colonies were checked for inserts by PCR. All analyzed colonies from 4- or 5-day-old cultures had an insert demonstrating the suitability of the selection regime.

In total, 97 brownish colonies were obtained which carried a DNA insert (confirmed by PCR) upstream of the pyrBC-lacZ fusion in pITM3_pyrB. These transconjugants were subsequently cultivated in f/2 GLUT solid medium with or without uracil to confirm their auxotrophy in culture media. From these 97 transconjugants, 23 cultures did not show the expected lack of growth suggesting a false-positive result. The PCR products for the remaining 74 colonies were sequenced. Nucleotide sequence analysis indicated that almost one third of the sequences occurred in duplicates so that a total of 41 individual and unique insert DNAs could be obtained. The corresponding IVET plasmids were isolated from the transconjugants and used for further characterization.

Interestingly, from the 41 insert DNAs, 30 inserts were in the same 5′-3′ orientation in terms of their downstream reading frames and the pyrBC-lacZ fusion of the IVET plasmid, while 11 were found in the opposite orientation on the antisense strand of an annotated coding region. In addition to the in co-culture induced clones, a randomly chosen blue colony representing a constitutively expressed pyrBC-lacZ phenotype was sequenced and used as a control in the following experiments. The insert of this transconjugant showed sequence identities to the upstream sequence of a gene encoding aldehyde dehydrogenase (Locus tag: HP15_943).

To confirm that the 30 cloned insert sequences were specifically allowing expression of pyrBC-lacZ during the interaction with T. weissflogii, the growth patterns of 13 randomly chosen in vitro uracil-deficient brown transconjugants (colonies 1, 10, 15, 22, 26, 28, 30, 68, 90, 100, 161, 168, and 194), and the blue transconjugant were analyzed in individual co-cultures with the diatom in f/2 medium without uracil (Figure 3). A transconjugant carrying the empty IVET plasmid was used as a negative control. All 13 positive transconjugants were able to survive the diatom co-incubation reaching cell numbers comparable to that of the wild-type or the blue transconjugant, which harbors a constitutively expressed promoter sequence. After 4 days of incubation, there was a more than three orders of magnitude difference in viable cell numbers of the positive clones and the negative control demonstrating that the identified promoter sequences were induced and enabled the ΔpyrB mutant to survive during the interaction with T. weissflogii.

In order to rule out any possible genomic alterations in the 41 mutant transconjugants, re-isolated IVET plasmids were individually re-introduced to the ΔpyrB mutant for a second time. Novel transconjugants were again tested for growth in f/2 GLUT solid medium with or without uracil but with X-Gal: These novel transconjugants did not grow when uracil was missing but grew well on uracil-containing medium indicating that the corresponding promoter sequences were not active and that the mutant transconjugants remained auxotrophic in vitro (Data not shown). Colonies on agar plates with uracil were brownish in color just like those of the wild type of HP15. While this result was expected for the 30 IVET plasmids, which contained promoter sequences in the same orientation as the annotated downstream gene, the corrrspondingly positive results for the remaining 11 IVET plasmid with DNA sequences in opposite direction to annotated genes remained obscure. One can only speculate that the automatic annotation of the HP15 genome (accession no. CP001978.1) might still be partially incomplete or incorrect (Gärdes et al., 2010).

M. adhaerens HP15 genes under the control of the promoters identified as specifically induced during interaction with T. weissflogii are listed in Table 3. All accession numbers are from Genbank entries CP001978.1 (M. adhaerens HP15 genome) or CP001978.1 (M. adhaerens HP15 187-kb indigenous plasmid). The possible functions of the correspondingly down-stream located genes were deduced by analyzing its amino acid sequences and determining the presence of conserved domains or similarities with reported proteins in the GenBank. Genes identified in the 11 transcriptional fusions orientated in the opposite direction to the in vitro reporter gene were not considered any further.

Detected genes were grouped depending on their potential ecological role during the interaction as follows: Genes that encode enzymes required in (i) detection of diatom cells; (ii) attachment to the diatom cells; (iii) putative metabolic exchange with diatom cells; and (iv) unknown functions (Table 3). Their potential role(s) will be discussed below.

Chemotaxis is a mechanism that bacteria use to chemically sense their environment and move toward attractants or away from repellents (Baker et al., 2005). In this process, chemoreceptors interact with specific chemicals—often amino acids—and interact with key chemotaxis proteins to control flagellar or pilus-mediated motility (Eisenbach, 1996). Chemoreceptors have a methyl-accepting chemotaxis domain, a homolog of which was found in the putative HP15 protein encoded by the gene downstream of the promoter sequence found in col. 178 (Table 3). Therefore, our current results support the previous finding that chemotaxis of HP15 is important for the diatom-bacteria interaction (Sonnenschein et al., 2012). Another example supporting this finding is col. 204 representing a genetic locus encoding a response regulator containing a receiver domain (HP15_3664) and a histidine kinase (HP15_3665). These proteins belong to the two-component signal transduction systems used by bacteria to respond to different stimuli and to regulate diverse cellular processes (Capra and Laub, 2012). They could be involved in chemotaxis since HP15_3664 shares similarities to chemotaxis-associated CheY-like response regulators (Stock et al., 1990). With new elegant technical approaches now available (Lambert et al., 2017) and our mutant arsenal in the model organism M. adhaerens HP15 (Sonnenschein et al., 2012), it is planned to investigate in depth the chemotactic behavior of HP15 in future studies.

An example of a regulatory gene potentially involved in sensing the diatom was found in col. 1 encoding the MerR1 transcriptional regulator (HP15_158) which has been described to mediate responses to environmental stimuli such as heavy metals, drugs, antibiotics, or oxidative stress (Brown et al., 2003). During the diatom's photosynthesis or photolysis of dissolved organic matter many reactive oxygen species are released (Asada, 2006). The ability to detoxify reactive oxygen species represents a selective advantage in the aquatic environment (Glaeser et al., 2010). Hünken et al. (2008) had shown that certain diatom-interacting bacteria protect the diatom by detoxifying oxygen species produced during the diatom's growth. However, it remains to be tested in future studies whether MerR1 is important for sensing reactive oxygen species or other potential threads such a heavy metals. The later possibility is supported by the IVET clone no. 30 containing the sequence of a genetic locus encoding the outer membrane component CzcC (HP15_108) of the heavy metal efflux pump CzcCBA (Nies, 2003). Interestingly, M. adhaerens HP15 possesses two highly homologous copies of fully functional czcCBA clusters prompting speculations about its pronounced role in detoxification processes (Stahl et al., 2015) and suggesting future studies on these clusters.

To initiate a close interaction, bacteria have to attach to diatom cells. The expression of genes, which change the adhesiveness of bacterial surface and promote adhesion has been previously observed in bacterial communities associated to algal blooms by meta-transcriptomics (Rinta-Kanto et al., 2012). In addition, proteins playing a role in biofilm formation have been identified in bacterial cultures growing with diatoms (Bruckner et al., 2011). Herein, we identified two genes and one operon that might be involved in the attachment of HP15 to T. weissflogii. Firstly, the gene associated with col. 32 was annotated to encode a protein with a putative conserved bacterial Ig-like domain. Proteins carrying this domain were found to be important in host-pathogen interactions (Kelly et al., 1999; Raman et al., 2010). For example, this domain is present in intimin, an outer membrane adhesion protein present in enteropathogenic or enterohaemorrhagic E. coli (Frankel et al., 2001). Secondly, the genetic locus identified in col. 168 encodes the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) IIA-like nitrogen-regulatory protein (Cases et al., 2001). The PTS system consists of a group of phosphotransfer proteins involved in the transport of carbohydrates, in chemotaxis toward carbon sources, and in the regulation of other metabolic pathways (Postma et al., 1993). A similar PTS system, was previously identified by IVET in Erwinia chrysanthemi interacting with tomato plants (Yang et al., 2004). Mutation of this gene affected the systematic invasion capability of the plant pathogen suggesting that certain PTS systems might be pivotal for the attachment of bacteria with photosynthetic eukaryotic cells.

The third genetic locus associated with attachment is the most exciting finding: In col. 68, a plasmid-borne sequence located upstream of the gene encoding Type IV fimbrial low-molecular weight Flp pilin (Kachlany et al., 2000) was identified suggesting that expression of a corresponding pilus might be diatom-inducible. This gene is the first of the so-called tight adherence (tad) locus. The tad locus was described in a wide range of pathogenic and non-pathogenic bacteria as well as in archaea, where it plays an important role in the colonization of respective, surprisingly diverse environmental niches (Kachlany et al., 2001). In Pseudomonas aeruginosa, Flp pili contribute to adherence to abiotic surfaces and surfaces of eukaryotic cells (de Bentzmann et al., 2006). A homologous locus was also identified in Caulobacter crescentus and designated cpa (Caulobacter pilus assembly) (Bodenmiller et al., 2004). Due to its wide-spread distribution and presence on a mobile genomic island the tad locus was termed Widespread Colonization Island (WCI) (Planet et al., 2003). In the human peridontal pathogen, Aggregatibacter actinomycetemcomitans this locus is composed of the genes flp1–flp2–tadV–rcpCAB–tadZABCDEFG encoding proteins that constitute a fibril secretion system (Kachlany et al., 2000). This operon is responsible for tight adherence, autoaggregation, biofilm formation, and the production of bundled fimbria-like fibers of individual pili (Kachlany et al., 2001; Planet et al., 2003; Perez et al., 2006; Tomich et al., 2007). Interestingly, our model organism, HP15 possess the entire WCI allowing the cautious suggestion that this organism might employ similar mechanisms of attachment to diatom surfaces as pathogens use for their host cell surfaces. Future studies should focus, among others, on determining the specificity of the corresponding pili for a given attachment surface thus shedding light on role of the diatom cell wall for this interaction.

Diatoms secrete organic compounds in the form of DOC or extracellular polymeric substances (Passow, 2002), which are taken up by heterotrophic bacteria (Grossart et al., 2006a). Gene expression analysis has identified an increase of transcripts involved in DOC uptake when bacteria interact with algae, e.g., transport systems for amino acid, carboxylic acids, carbohydrates (Poretsky et al., 2005, 2010; Rinta-Kanto et al., 2012; Teeling et al., 2012; Durham et al., 2017). In the current study, several promoters driving the expression of genes involved in the uptake of organic—mainly nitrogen-containing—compounds were identified. In col. 10 a genetic locus was identified which showed high sequence similarities to the codBA operon of E. coli previously described to have a putative role in a salvage pathway for nucleobases and being inducible by nucleobase starvation (Danielsen et al., 1992). Since the HP15 ΔpyrB mutant is deficient in pyrimidines biosynthesis and there is no pyrimidine available during its growth with T. weissflogii, the first explanation for the expression of this operon might be to allow the bacteria to survive under such conditions. However, DNA uptake by heterotrophic marine bacteria has been previously reported playing an important role as a supplement for nutrients or for new DNA synthesis (Jørgensen and Jacobsena, 1996).

The gene trapped in col. 211 encodes a protein containing a major facilitator superfamily (MFS) domain. Proteins containing this domain are secondary carriers that function as transporters for small solutes, such as monosaccharides, disaccharides, peptides, amino acids, and other molecules (Pao et al., 1998). Our result thus suggested that uptake of such compounds might be induced during the interaction. Similarly, gene ybgC (found in col. 161) encodes a homolog of the Tol-Pal system-associated acyl-CoA thioesterase, the first enzyme in the Tol-Pal system of E. coli (Godlewska et al., 2009). This system consists of five proteins (TolQ, TolR, TolA, TolB, and Pal) forming a membrane associated complex, which maintains outer membrane integrity. In addition, the operon shows similarity to the TonB system, which is important for active transport of diverse substrates such as siderophores or vitamin B12 (Moeck and Coulton, 1998) indicating that HP15 might possibly use TonB-dependent outer membrane receptors for uptake of yet-to-be determined substances. In their seminal paper, Teeling et al. (2012) had pointed out that TonB-dependent transport mechanisms were instrumental for marine bacterial population during algal bloom in the German bight.

Once the yet-unknown organic compounds released by the diatom are transported into the bacterial cells, they may be (partially or fully) degraded. In this context, we identified six genetic loci, which unfortunately do not allow concluding anything directly in terms of increased catabolism or accelerated energy conservation during the co-culture (col. 15, 19, 24, 149, 205, and 240). However, these results confirmed previous metagenomic and transcriptomic studies in which genes for degradation of organic compounds and an increase in bacterial activity and cell division rates were identified during bacteria-algae interactions (Poretsky et al., 2010; Smith et al., 2013; Durham et al., 2017).

In seven of the identified transconjugants, the IVET plasmids contained upstream sequences of genes involved in amino acid uptake (col. 26, 65, and 100), amino acid or protein degradation (col. 238 and 239), or even amino acid biosynthesis (col. 54 and 165). Inserts of two additional clones physically clustered with those of col. 54 and 238 but indicated sequence similarities to conserved hypothetical proteins (col. 166 and 241). This over-proportional appearance of amino acid-associated gene products among in co-culture induced proteins prompts the assumption that HP15 might preferentially obtain and use amino acids or amino group containing substances derived from the diatom cells. A somewhat similar observation was made by Durham et al. (2017), who found that presence of T. pseudonana induced expression of uptake systems for organic nitrogen compounds in the marine bacterium Ruegeria pomeroyi DSS-3. Our previous proteomics approach combined with a functional metabolite analysis (Stahl and Ullrich, 2016) had also revealed that M. adhaerens HP15 utilizes amino acids but not sugars as preferred carbon sources.

While the co-occurrence of genetic loci encoding amino acid-degrading and amino acid-synthesizing enzymes is a bit puzzling to interpret, the simultaneous finding of three amino acid or amino compound uptake systems being required during the interaction seems to be promising: The genetic locus HP15_3541 (col. 65) encodes for the periplasmic portion of a spermidine/putrescine transporter. Diatom cell walls contain high amounts of long-chain polyamines, which are organic constituents of diatom bio-silica (Kröger et al., 2000; Kröger and Poulsen, 2008). Previously, a positive correlation was found between the concentrations of polyamines in seawater and the amount of transcripts from different groups of marine bacteria involved in the degradation of polyamines (Mou et al., 2011). Transcripts and genetic evidence involved in polyamines uptake were also found in bacteria associated to marine phytoplankton blooms (Moran et al., 2004; Poretsky et al., 2005, 2010) as well as in free-living freshwater bacteria (Garcia et al., 2013). Our data thus allowed us to speculate on a potential bacterial use of polyamines shed during diatom cell wall synthesis and prompts future experiments in this direction.

The IVET screen revealed two further genetic loci (col. 100 and 26), whose expression was associated with presence of T. weissflogii, and which encode for LivK (HP15_4098) and AotJ (HP15_3031), respectively. Interestingly, the gene product of livK was already found upregulated in our previous proteomics study (Stahl and Ullrich, 2016). LivK is part of a branched amino acid uptake system transporting leucine, isoleucine, and valine (Adams et al., 1990; Ribardo and Hendrixson, 2011). LivK's functional involvement in branched amino acid transport in M. adhaerens HP15 is supported by the close proximity of genes encoding the inner membrane translocators LivH and LivG as well as the ATP binding components LivM and LivF (Adams et al., 1990). Interestingly and in agreement with our finding, the RNAseq-based transcriptome analysis of R. pomeroyi DSS-3 revealed other branched amino acid uptake systems being upregulated in presence of diatom cells (Durham et al., 2017). Since the later study found several additional bacterial transport systems to be diatom-induced, the observed functional analogy of branched amino acid uptake systems in both organisms might reflect both, the multifactorial nature of the respective interactions and possible common metabolic interaction themes.

AotJ is an amino acid-binding protein, which belongs to the arginine/ornithine uptake system (Wissenbach et al., 1995; Nishijyo et al., 1998). AotJ's potential function is substantiated by presence of surrounding genes encoding the subunit of a histidine-lysine-arginine-ornithine transporter and two inner membrane permeases of arginine and histidine associated ABC transporters in the genome of HP15. Just like LivK, the AotJ gene product was found upregulated in M. adhaerens HP15 during exposure to T. weissflogii cells in our previous proteomics approach (Stahl and Ullrich, 2016). Both proteomics hits now substantiated by the IVET screen may hint at an important role of bacterial amino acid uptake during the bacteria-diatom interactions with a distinct focus on branched chain amino acids and arginine/ornithine, respectively.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.