Globaalse regulaatorvalgu Fis-i roll lapF geeni ekspressiooni reguleerimisel ja rakupinna hüdrofoobsuse mõjutamisel mullabakteris Pseudomonas putida

Väitekirja elektrooniline versioon ei sisalda publikatsiooneBakteritel on pikaaegse evolutsiooni käigus välja kujunenud omadused ja mehhanismid selleks, et olla kohanemisvõimelised ja jääda ellu mitmesugustes keskkonnatingimustes. Vastavalt vajadusele muudavad bakterid oma eluvormi, ujudes vabalt keskkonnas ringi või olles paiksed ja moodustades koos teiste bakteritega ühise kooseluvormi, mida nimetatakse ka biofilmiks. Biofilmis elades on bakterid paremini kaitstud kahjulike keskkonnamõjude eest. Üleminek liikuvalt eluviisilt paiksele algab tavaliselt bakterite kinnitumisega elusale või elutule pinnale. Erinevatele pindadele kinnitudes võivad bakterid tekitada tõsist kahju näiteks meditsiini ja tööstusvaldkondades. Samas, biofilmi moodustavaid baktereid on kasutatud ka edukalt näiteks mürgiste ühendite eemaldamiseks keskkonnast. Üks peamisi bakteriraku pinna omadusi, mis aitab kinnitumisele kaasa, on hüdrofoobsus. Hüdrofoobsema pinnaga rakud liituvad ehk agregeeruvad omavahel paremini, tänu millele nad vähendavad kättesaadavat rakupinda kahjulikele ainetele. Näiteks mullabakter Pseudomonas putida muudab oma rakupinda hüdrofoobsemaks kokkupuutel erinevate toksiliste ainetega. P. putida on kosmopoliitne bakter, mis tihti elab taimejuurtel ja soodustab taimede kasvu. Üldiselt mullabakterid eelistavad elada taimejuurte ümber risosfääris, sest taimed eritavad juurte kaudu bakterite jaoks kergesti kasutatavaid toitaineid. Samas, taimed võivad juurte kaudu ümbritsevasse mulda eritada ka toksilisi ühendeid. Seega bakterid peavad kohanema nii kasvu soodustavate kui ka pärssivate tingimustega. Käesolevas töös kirjeldatakse P. putida pinna hüdrofoobsufaktorit LapF-i ning selle geeni regulatsiooni Fis-ga. Varem ei ole avaldatud seoseid LapF-i ja P. putida pinna hüdrofoobsuse vahel ega ole ka teada detailsemat infot lapF geeni ekspressiooni regulatsiooni kohta. P. putida rakud, mis toodavad pinnavalku LapF, on stressitingimustes hüdrofoobsemad võrreldes rakkudega, kus puudub LapF. LapF-st tulenev hüdrofoobsem rakupind võib omada potentsiaalset kaitsvat rolli P. putida’le. LapF-ga P. putida’l on soodustatud rakkude liitumine, tänu millele vähendatakse toksilistele ainetele kättesaadavat rakupinda. Kirjeldatud kaudne kaitsemehhanism võib olla üks seni avastamata LapF-i rolle mullabakteris P. putida.Bacteria have developed several mechanisms to cope with the changing conditions of the surrounding environment. The characteristics of cell surface can passively help to deal with unfavourable conditions or can be involved in the switch between two different life-forms: free-swimming aka planktonic lifestyle and surface-attached aka biofilm aka sessile lifestyle. When living in a biofilm, bacteria are better protected against harmful conditions. The transition from free-swimming to sessile happens when bacteria attach to the surface and/or each-other. The formation of bacterial biofilms can cause serious problems for humans. Hospital-related infections due to bacteria on surgical instruments are one of the examples. However, the human society has also learned to benefit from the bacterial biofilm by using microorganisms to remove contaminants from environment. In most cases, the adhesion properties of bacteria are defined by cell surface hydrophobicity. Bacteria with more hydrophobic surface attach to each-other, which helps reduce their surface to toxic compounds. For example, the surface of Pseudomonas putida becomes more hydrophobic in the presence of toxic chemicals. P. putida is a soil bacterium mainly residing on the roots of plants. That kind of habitation is strongly dependent on the roots exudates, which include both nutrients and toxic compounds for bacteria. In order to survive and outcompete other microorganisms on the plant roots P. putida must be able to quickly adapt to the changes in its surrounding environment. For example staying put by forming biofilm or moving freely to recolonize new parts of plant roots for better conditions. The aim of this research was to study and enlighten the role of surface protein LapF in P. putida and it`s regulation by global regulator Fis. Prior this work, LapF had only been described as a biofilm-related protein and there was no detailed information published about the lapF gene regulation in P. putida. We demonstrate, that LapF is the factor for making P. putida cells more hydrophobic and propose that LapF might have a potential role as a protective factor for P. putida. More hydrophobic bacteria can be more aggregative and therefore might help the cells reduce the surface exposed to the environment. This can be a passive protection mechanism against toxic compounds

Pseudomonas putida Fis binds to the lapF promoter in vitro and represses the expression of LapF.

The biofilm matrix of the rhizospheric bacterium Pseudomonas putida consists mainly of a proteinaceous component. The two largest P. putida proteins, adhesins LapA and LapF, are involved in biofilm development but prevail in different developmental stages of the biofilm matrix. LapA is abundant in the initial stage of biofilm formation whereas LapF is found in the mature biofilm. Although the transcriptional regulation of the adhesins is not exhaustively studied, some factors that can be involved in their regulation have been described. For example, RpoS, the major stress response sigma factor, activates, and Fis represses LapF expression. This study focused on the LapF expression control by Fis. Indeed, using DNase I footprint analysis a Fis binding site Fis-F2 was located 150 bp upstream of the lapF gene coding sequence. The mapped 5' end of the lapF mRNA localized the promoter to the same region, overlapping with the Fis binding site Fis-F2. Monitoring the lapF promoter activity by a β-galactosidase assay revealed that Fis overexpression causes a 4-fold decrease in the transcriptional activity. Furthermore, mutations that diminished Fis binding to the Fis-F2 site abolished the repression of the lapF promoter. Thus, these data suggest that Fis is involved in the biofilm regulation via repression of LapF expression

Effect of overexpression of Fis on the level of transcription from the <i>lapF</i> promoter.

<p>β-Galactosidase (β-Gal) activity expressed from the <i>lapF</i> promoter-lacZ reporter constructs were measured in the wild-type strain PSm and the Fis-overexpressing strain F15 of <i>P. putida</i> grown for 18 hours in LB medium supplemented with 1 mM IPTG and without IPTG supplementation. Vertical bars denote 95% confidence intervals of means. Data of at least 8 independent measurements is shown. The length of the <i>lapF</i> promoter region inserted upstream of the <i>lacZ</i> reporter gene in different pBLKT constructs is shown above diagrams. The Fis-binding site Fis-F2 (gray box) and 5′ end of <i>lapF</i> mRNA F-I (arrow) are shown in drawings.</p

Gel shift assay of the Fis binding to the <i>lapF</i> promoter DNA containing the wild-type Fis binding site Fis-F2 and the mutated site Fis-F2-mut.

<p>2×10¹⁰ molecules of radioactively labelled PCR products containing Fis-F2 (lanes 1–10) and Fis-F2-mut site (lanes 11–20) were used for Fis binding. Fis was outcompeted from Fis-DNA complex with unlabelled PCR product containing the Fis binding site (LF2) and PCR product without Fis-binding site (RF1). Added unlabelled DNA was calculated in molecules. 0.46 µM Fis was used in each reaction mixture except mixtures without Fis in lanes 2 and 12.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida.

The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida

Fis binding to Fis-A1 and Fis-A2 sites upstream of the <i>lapA</i> gene.

<p>(A) Protection of the <i>lapA</i> upstream DNA against DNase I cleavage by Fis binding on the sense and the antisense strands. Lines at the right side of the panels indicate the regions protected by Fis from DNase I cleavage at the positions -110 to -85 on the sense strand and -109 to -84 on the antisense strand corresponding to Fis-A1; and -158 to -132 on the sense strand and -159 to -138 on the antisense strand corresponding to Fis-A2. (B) Gel shift assay of the Fis binding to the <i>lapA</i> promoter DNA containing the wild-type Fis binding site Fis-A1 and Fis-A2 and one or the other mutated site. 2 × 10¹⁰ molecules of radioactively labelled PCR products containing both Fis binding sites Fis-A1 and FisA2 or one mutated binding site FisA1mut-Fis-A2, and Fis-A1-FisA2mut were used in Fis binding assay. Fis was outcompeted from Fis-DNA complex with unlabelled PCR product containing the Fis binding site (LF2) or a PCR product without Fis binding site (RF1). Arrows point to different dissociation of Fis from radioactively labelled DNA in favour of binding unlabelled Fis-specific DNA. Added unlabelled DNA was calculated in molecules. 0.46 μM Fis was used in each reaction mixture except mixtures without Fis in lanes 2, 12 and 22.</p

The effect of mutated Fis binding sites Fis-A6 and Fis-A7 to P_<i>lapA8</i> promoter.

<p>B-galactosidase (β-Gal) activity expressed from the <i>lapA</i> promoter-<i>lacZ</i> reporter constructs p9_P_lapA8B, p9_P_lapA8B_F6mut, p9_P_lapA8B_F7mut and p9_P_lapA8B_F6,7mut were measured in <i>P</i>. <i>putida</i> wild-type strain PSm and <i>fis</i> overexpression strain F15 grown in LB medium with or without 1 mM IPTG for 18 hours. Schemes of Fis binding sites (shown as grey boxes) are shown below the diagrams. Dotted lines denote mutated Fis binding sites, <i>lacZ</i> reporter gene is shown as a black arrow and promoter P_<i>lapA8</i> in a small white box. The scheme is not to scale. Vertical bars denote 95% confidence intervals of means. Data of at least 9 independent measurements is shown. Letters a–i depict homogeneity groups according to ANOVA <i>post hoc</i> Bonferroni test. Identical letters denote non-significant differences (<i>P</i>>0.05) between averages of β-galactosidase activity.</p