Microbiology Papers in Press. Published July 10, 2014 as doi:10.1099/mic.0.075317-0

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TITLE PAGE

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Word count of the manuscript: 4366

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Journal's contents category: Environmental and Evolutionary Microbiology

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Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains

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under aerobic and anaerobic conditions

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Suelen B. Costa1, Ana Carolina C. Campos1, Ana Claudia M. Pereira1, Ana Luiza de

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Mattos-Guaraldi1, Raphael Hirata Júnior1, Ana Cláudia P. Rosa1 and Lídia M. B. O.

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Asad2*

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Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Ciências

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Médicas, Universidade do Estado do Rio de Janeiro 2

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Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro

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Running Head: Role of BER in adherence and filamentation of E. coli.

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*

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Departamento de Biofísica e Biometria, IBRAG, UERJ. Av. 28 de Setembro, 87 –

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Fundos, 4o andar. Vila Isabel, RJ, Brazil. CEP: 20.551-030

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Tel +55 (021) 2587-6507; FAX +55 (021) 2587-6476

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E-mail: [email protected]

Correspondence: Lídia Maria Buarque de Oliveira Asad

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Abstract

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During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-

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damaging agents and these agents can induce several defense mechanisms. Base

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excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by

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reactive oxygen species (ROS) generated by chemical and physical agents or by

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metabolism. In this work, we have evaluated whether the interaction with an abiotic

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surface by mutants derived from Escherichia coli K-12 deficient in some enzymes that

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are part of BER causes DNA damage and associated filamentation. Moreover, we

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studied the role of endonulease V (nfi gene; 1506 mutant strain) in biofilm formation.

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The endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions.

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We verified that endonuclease V is involved in biofilm formation. Our results showed

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more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant

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(BW535) than in the wild-type strain (AB1157). In contrast, the mutant nfi did not

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present filamentation in biofilm, although its wild strain (1466) showed rare filaments in

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biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of

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SOS induction measured by the SOS-chromotest. However, biofilm formation depended

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on the ability of the bacteria to induce the SOS response since the mutant lexA Ind-

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doesn’t induce the SOS and doesn’t form any biofilm. The oxygen tension was an

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important factor for the interaction of the BER mutants, since these mutants exhibited

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decreased quantitative adherence under anaerobic conditions. Regardless of, nfi mutant

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and its wild type did not exhibited decreased in biofilm under anaerobic conditions.

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However, this decrease was not related to the BER mutants’ death. Scanning electron

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microscopy was also performed on the E. coli K-12 strains that had adhered to the glass,

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and we observed the presence of a structure similar to an extracellular matrix that

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depended on the oxygen tension. In conclusion, it was proven that bacterial interaction

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with abiotic surfaces can lead to SOS induction and associated filamentation. Moreover,

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we verified that the endonuclease V is involved in biofilm formation.

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Keywords: Reactive oxygen species (ROS). Base excision repair (BER). SOS system.

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F3ilamentation. Biofilms.

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3

1 2 3

Introduction Escherichia coli is a predominant species among facultative anaerobic bacteria

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of the gastrointestinal tract. Both its frequent community lifestyle and the availability of

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a wide array of genetic tools contributed to establish E. coli as a relevant model

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organism for the study of surface colonization (Beloin et al., 2008).

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Bacterial adherence is a key step in bacterial physiology and pathogenesis and this

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adherence to biotic and abiotic surfaces is a complex process that, in many cases,

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involves the participation of several distinct adhesins, all of which may act at the same

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time or at different stages during colonization (Ofek et al., 2002; Rendón et al., 2007).

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During colonization, the bacteria often encounter DNA-damaging agents and these

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agents can induce various defense mechanisms.

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The SOS system in E. coli is regulated by LexA–RecA repressor-activator

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proteins, respectively. In the E. coli SOS response, the expression of approximately 40

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unlinked genes is induced after the cell is exposed to DNA-damaging agents (Fernandez

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et al., 2000; Friedberg et al., 2006). Many of these gene products are involved in

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chromosome recombination, replication, repair, and segregation during cell division

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(Friedberg et al., 2006; Gotoh et al., 2010). Filamentation is a consequence of the SOS

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response. During SOS, premature cell division is prevented, and genes for DNA damage

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repair are induced. SulA, the SOS gene product, inhibits cell division by binding to FtsZ

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to block septum formation until the DNA damage has been repaired (Higashitani et al.,

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1995; Gotoh et al., 2010). Although the filamentation protects daughter cells from

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receiving damaged copies of the bacterial chromosome, the filamentous phenotype can

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also involve other programmers that are designed to promote bacterial survival (Justice

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et al., 2008).

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Recently, it has been shown that the activation of the SOS response is involved in the

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adherence to abiotic surfaces by E. coli (Hoffman et al., 2005; Linares et al., 2006;

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Gotoh et al., 2010), Vibrio cholerae (Hung et al., 2006; Gotoh et al., 2010) and

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Mycobacterium avium (Geier et al., 2008; Gotoh et al., 2010).

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Aerobically grown cells are exposed to endogenous reactive oxygen species

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(ROS), including singlet oxygen, superoxide, hydrogen peroxide, and the hydroxyl

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radical. The ROS can damage intracellular components such as lipids, proteins, and

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DNA (Imlay, 2003; Sakai et al., 2006). The mutagenesis of DNA may be generated

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through spontaneous mutations. However, most organisms possess mechanisms to

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detoxify the ROS, removing the oxidative DNA damage (Sakai et al., 2006).

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Base excision repair (BER) is dedicated to the repair of the oxidative DNA

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damage caused by ROS generated by chemical and physical agents or by metabolism.

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BER involves two classes of enzymes, bifunctional glycosylases such as the

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endonuclease III encoded by nth gene, and apurinic/apyrimidinic (AP)-endonucleases,

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such as endonuclease IV and exonulease III, encoded by genes nfo and xth, respectively

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(Wallace, 1997). E. coli possesses a homologue of every BER enzyme identified (Eisen

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& Hanawalt, 1999). The redundancy in BER may provide evidence for its biological

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significance to E. coli strains.

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Endonuclease V plays the initiating role in base recognition and strand nicking.

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Because endonuclease nicks at the 3´ side of the lesion and the base damage is not

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removed from DNA. Other enzymes have to act to remove nicks in the 3 'site of the

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lesion. So, several proposals have been put forward to explain the removal of the

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damage base by 3`exonuclease or endonuclease action (Cao, 2013).

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Defense mechanisms invoked against the oxidative stress can be involved in

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various bacterial responses such as antibiotic resistance (Kohanski et al., 2007), increase

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of survival within macrophages (Suvarnapunya et al., 2003), and the production of

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diversity and adaptability in biofilm communities (Boles & Singh, 2008).

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The essential role of oxygen in biofilm formation is controversial since Colón-

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González et al., 2004, reported that E. coli K-12 strains are unable to form biofilms

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under anaerobic conditions and Bjergbaek et al., 2007, reported that E. coli K-12 strains

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had the ability to form biofilm in the absence of oxygen.

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In this paper, we evaluated whether the adherence to abiotic surfaces would be

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able to generate genotoxic effects in mutants deficient in BER that were derived from E.

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coli K-12 with induction of the SOS system and associated filamentation. The current

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study showed that the filamentation observed was a consequence of the SOS induction,

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triggered by attachment to the glass surface. In part, the filamentation and SOS

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induction observed in these experiments were related to ROS production, since both

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decrease of filamentous growth and SOS response occurs even when the assays of

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adherence were performed under anaerobic conditions.

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Moreover, we verified the participation of endonuclease V in biofilm formation

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in both aerobic and anaerobic conditions once this enzyme has a significant role in the

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repair of base damage such as BER mutants.

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1 2

Materials and Methods

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Bacterial strains.

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The bacterial strains derived from E. coli K-12 used in this study are listed in

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Table 1. All strains are isogenic, except for the particular mutation of interest. The

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prototype 042 EAEC strain (AAF/II), originally isolated from an infant with diarrhea in

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Lima, Peru (Nataro et al., 1985) was used as a positive control. E. coli K-12 DH5α

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strain was used as negative control in all assays. Microorganisms were stored at -70ºC

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in Luria broth (LB, Merck, S.A.) with 20% glycerol.

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Bacterial culture conditions.

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Biofilm formation assays were done with bacterial strains grown in LB, 18 h at

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37ºC, under aerobic and anaerobic conditions. The anaerobic conditions were generated

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by an AnaeroGen kit (Oxoid).

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Biofilm methods

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To assess qualitative biofilm formation, 1 ml of Dulbecco’s modified Eagle’s

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medium (DMEM) containing 0.45% glucose was added to each well of 24-well tissue

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culture plates with circular glass cover slips (Nunc International, USA). Then, 10 μl

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(approximately 107 bacterial cells) of bacterial cultures grown overnight in LB, under

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aerobic and anaerobic conditions were incubated with DMEM 18 h at 37ºC, under

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aerobic and anaerobic conditions. At the end of the incubation time, the culture medium

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was aspirated, and the substratum was washed three times with Dulbecco’s phosphate

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buffered saline (PBS-D), fixed with methanol, and stained with 0.5% crystal violet (CV)

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for 5 min. The stained coverslips were mounted on glass slides and examined by light

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microscopy (Sheikh et al., 2001).

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To assess semi-quantitative biofilm formation, 200 μl of DMEM containing

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0.45% glucose in a 96-well flat-bottom polystyrene microtiter plate (TPK) was

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inoculated with 5 μl (approximately 105 bacterial cells) of a culture of LB grown

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overnight at 37ºC, under aerobic and anaerobic conditions. The plate was incubated for

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18 h at 37ºC, under aerobic and anaerobic conditions. Then, the planktonic cells were

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removed by rinsing three times with water, and the substratum was stained with 0.5%

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CV for 5 min. After washing with water and blotting with paper towels, the biofilm

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formation was quantified spectrophotometrically by the addition of 200 μl of 95%

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ethanol to each well of a CV-stained microtiter plate. After solubilization for 30 min at

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room temperature, 150 μl was transferred to a new microtiter plate, and the absorbance

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was determined with an enzyme-linked immunosorbent assay (ELISA) plate reader at

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570 nm (Mohamed et al., 2007).

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Biofilm viability

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Biofilm viability was evaluated by a FilmTracer live/dead biofilm viability Kit

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(Invitrogen). The qualitative biofilm formation was performed as previously described

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in biofilm methods with some modifications. After an 18 h incubation period at 37ºC,

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under aerobic or anaerobic conditions, the substratum was washed with water and

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stained with 200 μl of staining solution (1μl of SYTO 9 stain and 1 μl of propidium

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iodide stain to 1 ml of filter-sterilized water) onto the biofilm sample for 30 min, at

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room temperature, protected from light. After washing with water, the stained coverslips

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were mounted on glass slides and examined by confocal microscopy.

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SOS induction.

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The bacterial strain PQ35 was cultured in 5 ml of LB containing 50 μg ml-1

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ampicillin for 18 h at 37ºC, under aerobic and anaerobic conditions. Then the cells were

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centrifuged, re-suspended in 1 ml PBS-D, and 10 μl (approximately 108 bacterial cells)

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of the bacterial suspension was added to 24-well tissue culture plates with glass slides,

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and DMEM containing 0.45% glucose was added. After an 18 h incubation period,

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under aerobic or anaerobic conditions, the substratum was washed twice with PBS-D

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and overlaid with 1 ml of 1% Triton X-100 in PBS-D for 30 min. Samples of bacterial

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cells were removed of biofilm, centrifuged, and re-suspended in a saline buffer (Alves

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et al, 2010). The induction of the SOS system was measured in E. coli PQ35 (adherence

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to glass and planktonic cells) by means of a sfi::lacZ operon fusion according to the

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principle of the SOS Chromotest (Quilardet & Hofnung, 1985; Asad et al., 1997). The

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measurements of β –galactosidase and alkaline phosphatase activities were performed

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as previously described (Quillardet & Hofnung, 1985; Asad et al., 1997). The results

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were calculated according to the ratio R of β–galactosidase units (B) to alkaline

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phospatase units (P)

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(R = Abs420nmB X incubation time, in min/Abs420nmP X incubation time, in min)

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(Quillardet & Hofnung, 1985; Asad et al., 1997).

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Scanning electron microscopy.

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For scanning electron microscopy (SEM), biofilm specimens (18 h) obtained

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from glass slides were fixed in 2.5 % glutaraldehyde, post-fixed in 1 % osmium

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tetroxide, and dehydrated in a graded series of ethanol. Subsequently, the samples were

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subjected to critical point drying with carbon dioxide, covered with gold–palladium to a

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10 nm layer and examined with a JEOL JSM 5310 scanning electron microscope.

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Statistical analysis.

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All assays were performed three times under each condition in duplicate. The

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means from each experiment were analyzed by the Student´s t-test using the GraphPad

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Prism statistical software (GraphPad Software, Inc., CA, USA). Results were

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considered statistically significant when P ≤ 0.05.

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1 2

Results

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Expression of filamentous phenotype during adherence of bacterial strains to

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abiotic surfaces

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We previously showed that adherence to HEp-2 cells is accompanied by

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filamentous growth and SOS induction in wild type strain (AB1157), and especially

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BER mutants (BW9091 and BW535) (Costa et al., 2012). Therefore, we wondered

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whether the attachment to glass could lead to DNA damage in the same BER mutants

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resulting in a filamentation response.

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Microscopic inspection of bacterial adherence to glass after 18 h of incubation

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revealed that all strains of E. coli K-12 tested were able to adhere to glass (Fig. 1, Table

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2).

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Moreover, this adherence to glass was accompanied by filamentous bacterial

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growth in the xthA mutant (BW9091) (Fig. 1(b)) and especially in the triple xthA nfo nth

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mutant (BW535) (Fig. 1(c)). This result is similar to what was observed in the

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adherence to HEp-2 cells where BER mutants (BW9091 and BW535) also revealed

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higher filamentation (Costa et al., 2012). Nevertheless, the wild type strain (AB1157)

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presented rare filaments (Fig. 1(a)) and the strain deficient in SOS induction (lexAInd-

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DM49) did not exhibit bacterial cell filamentation (Fig. 1(d)). The nfi mutant (1506)

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also does not showed filaments such as lexAInd- different of its wild strain (1466) which

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showed rare filaments (Table 2).

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1 2

The influence of oxygen tension on filamentous growth during the adherence to

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glass of E. coli K-12 strains

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To examine the involvement of ROS in the filamentation of strains adhering to

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glass, we compared the filamentation of E. coli cells grown and attached to glass under

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anaerobic or aerobic conditions. The results displayed in Fig. 2 and Table 2 showed that

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all strains of E. coli K-12 tested were able to adhere to glass under anaerobic conditions

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(Fig. 2, Table 2). Furthermore, this adherence to glass is accompanied by a

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filamentation response even when the experiments were performed in the absence of

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oxygen, except for the mutant deficient in SOS induction (DM49) (Fig. 2) and nfi

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mutant that exhibited no filamentation when adhered to glass (Table 2).

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The SOS response expression after bacterial adherence to glass assays in the

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presence and absence of oxygen

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Filamentation is one of the characteristic features of the induction of the SOS

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response, which is triggered by DNA damaging agents (Walker, 1985). So we measured

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the SOS induction of recovered bacteria that adhered to glass and the bacterial cells

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grown in liquid media (DMEM), in the presence and absence of oxygen through an

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SOS-chromotest assay (Quillardet & Hofnung, 1985; Asad et al., 1997).

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In this assay the level of SOS induced was evaluated by measuring the induction

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of β-galactosidase in E. coli PQ35 (wild type strain), in which the lacZ gene has been

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fused to the sfiA gene, a SOS gene (Quilardet & Hofnung, 1985). We observed a higher

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β-galactosidase expression in the glass-attached bacteria when compared with DMEM-

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grown cells both under aerobic or under anaerobic conditions (Fig. 3). However, we

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observed a decrease in β-galactosidase expression in both glass-attached and DMEM-

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grown bacteria under anaerobic conditions (Fig. 3).

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Quantitative adherence to abiotic surface by E. coli K-12 strains in the presence

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and absence of oxygen

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The ability of E. coli K-12 strains to adhere to a different abiotic surface

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(polystyrene) was assessed by a quantitative test (Fig. 4). In this assay, it was observed

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that E. coli K-12 strains (AB1157, BW9091, BW535 and DM49) adhered less to

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polystyrene than the EAEC 042 strain did, which was used as a positive control.

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However, nfi mutant showed an increase in biofilm formation when compared with

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EAEC 042 strain under aerobic and anaerobic conditions (Fig. 5).

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Statistical analysis revealed a significant increase in adherence to polystyrene for

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the wild-type strain (AB1157) and BER mutants (BW9091 and BW535) as compared

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with the negative control E. coli K-12 DH5α. However, the lexA mutant deficient in

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SOS induction (DM49) did not present any significant difference in adherence to

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polystyrene in comparison with E. coli K-12 DH5α (Fig. 4).

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In aerobiosis, the mutants xthA (BW9091) adhered to the polystyrene substratum

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like the wild-type strain (AB1157) (Fig. 4). However, under the same conditions, the

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triple mutant BW535 (xthA nfo nth) presented a significant decrease in adherence to

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polystyrene when compared with the wild-type strain (AB1157) (Fig. 4).

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The oxygen tension affects the adherence of BER mutants (BW9091 and

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BW535) to polystyrene as compared with the wild-type strain (AB1157). Our results

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show that under anaerobic conditions the mutants xthA (BW9091) and the triple mutant

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BW535 (xthA nfo nth) present a significant decrease in adherence to polystyrene when

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compared with wild-type strain (AB1157) (Fig. 4). However, nfi mutant and its wild

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type strain did not show difference in biofilm formed under aerobic or anaerobic

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conditions (Fig. 5).

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The influence of oxygen tension on the viability of E. coli K-12 strains during

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adherence to glass

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To verify whether the reduction in the number of BER mutants adherent to

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polystyrene under anaerobic conditions was related to the death of the bacterial strains,

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viability tests were performed using the FilmTracer live/dead biofilm viability Kit.

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Thus, bacteria with intact cell membranes (i.e., live) would stain fluorescent green,

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whereas bacteria with damaged membranes (i.e., dead) would stain fluorescent red. Our

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results showed that the presence or absence of oxygen did not directly affect the

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viability of strains, since the number of viable bacteria observed was greater than the

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number of dead bacteria under aerobic as well as anaerobic conditions, for all strains

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tested (Fig.6).

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Scanning electron microscopy of E. coli K-12 strains adhered to glass

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Our results showed that the wild-type strain (AB1157) and xthA mutant

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(BW9091) presented microcolony formation on a glass surface surrounded by a

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structure suggestive of an extracellular matrix under aerobic conditions (Fig. 7(a), (c)).

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However, under anaerobic conditions the matrix-enclosed microcolonies were absent in

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both strains (Fig. 7(b), (d)). Moreover, the triple mutant xthA nfo nth (BW535) forms

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microcolonies on a glass surface, but did not present a structure suggestive of an

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extracellular matrix under aerobic or anaerobic conditions (Fig. 7(e), (f)). For this strain,

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intense filamentation was also observed under both conditions (Fig. 7 (e), (f)).

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Discussion

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We previously showed that induction of the SOS occurs when E. coli K-12

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strains interact with the epithelial cells (Costa et al., 2012). In this paper, we verified

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that adherence to abiotic surfaces is accompanied by filamentous growth. except for

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lexAInd- (DM49) and nfi (1506) mutant strains that did not induce SOS. Filamentation

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indicates the induction of the SOS system which was confirmed by SOS chromotest..

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Here it was observed that the SOS response was induced by the adhesion of bacteria to

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an abiotic surface.

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Recently, a clear link was shown between the activation of the SOS response

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and the adherence to abiotic surfaces by Listeria monocytogenes and Pseudomonas

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aeruginosa (van der Veen & Abee, 2010; Gotoh et al., 2010). Filamentation is a

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consequence of the SOS response. Clearly, the involvement of the SOS response in

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adherence to abiotic surfaces raises an interesting question about colonization surfaces

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(Gotoh et al., 2010).

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It is known that other systems inducible by ROS were not only involved in the

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defense against environmental stresses, but that they also participated in the adherence

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of E. coli to surfaces. For example, mutation of the E. coli oxidative stress response

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regulator gene oxyR results in the overexpression of an adhesin related to adherence to

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abiotic surfaces (Ag43). As a result, E. coli oxyR mutants display an increased

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adherence to abiotic surfaces and autoaggregation in addition to a decrease in motility

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(Ulett et al., 2006). Moreover, rpoS, another regulator gene involved in several stress

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responses, may affect cell adherence (Dong et al., 2009).

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Studies investigating the role of oxygen in bacterial interaction are of great

2

importance, because the primary niche of E. coli in vivo is the gastrointestinal tract

3

where conditions are anaerobic or of low oxygen tension (Bjergbaek et al., 2007).

4

Our results showed that under anaerobic conditions the bacterial strains tested

5

have the ability to adhere to glass and form filamentous growth. However, a reduction

6

in the filamentation was observed when wild-type strain (AB1157) and BER mutants

7

(BW9091 and BW535) were submitted to anaerobiosis. This reduction of filamentation

8

in the absence of oxygen was consistent with the decrease in SOS induction in this

9

condition, when compared to what was observed under aerobic conditions. These results

10

suggest that even under anaerobic conditions, other agents such as agents nitrous can be

11

produced which can be responsible for the DNA damage of these strains, inducing the

12

SOS and observable filamentation. The wild-type strain proficient in endonuclease V

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showed an decrease of filamentation in anaerobic conditions.

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The mutant nfi presented higher biofilm formation in aerobic or anaerobic

16

conditions. The endonuclease V is an enzyme that is involved in DNA repair of

17

nitrosative lesions (Weiss, 2001). Probably these lesions caused by nitrosating agents

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are generated independent of oxygen tension. Although, these agents are generated

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rather in anaerobic condition was observed increase of mutagenesis in nfi mutant in

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aerobic growth (Weiss, 2001). Because in nfi mutant the base damage is not removed

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from DNA and gaps are not formed, the SOS response is not induced and the

22

filamentation associated does not occur. However, wild type presented SOS response

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and filamentation.

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1

Paradoxically, the nfi mutant has an increased rate of survival when compared to

2

the wild strain. The increase of double DNA breaks in wild type strain is most lethal

3

than the unrepaired DNA mutagenic lesions in the nfi mutant (1506) (Guo and Weiss,

4

1998). This fact may explain the decrease in biofilm formation of the wild-type strain

5

(1466) compared to the nfi mutant.

6 7

We also measured the SOS induction of bacteria grown in liquid media

8

(DMEN), and a decrease of SOS response expression was observed in the presence or in

9

the absence of oxygen when compared with the SOS induction of bacteria adhered to

10

glass under both conditions. These results suggest that bacterial cells adhered to abiotic

11

surfaces, such as glass, present an increased SOS expression when compared to

12

planktonic form.

13

In order to quantify the adhesion to the abiotic surface, tests were performed

14

on another surface (polystyrene). Although the number of bacteria that attached to

15

polystyrene for each strain (AB1157, BW9091 and BW535) was less than what was

16

measured for the EAEC042 strain, which was used as a positive control in the test, it

17

was observed that the strains tested in this study adhered more than E. coli K-12 DH5α

18

(not forming biofilm), which was used as a negative control in this type of experiment

19

(Mohamed et al., 2007). This result indicates that these E. coli K-12 strains were able to

20

form biofilms under aerobic or anaerobic conditions. Our results also showed that the

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nfi mutant has a higher biofilm formation than the positive control EAEC 042 in aerobic

22

and anaerobic conditions.

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18

1

Gotoh et al., 2010, showed that the lexA mutant deficient in SOS induction did

2

not present any biofilm formation in Pseudomonas aeruginosa. Here it also was

3

observed that the lexA mutant deficient in SOS induction (DM49) did not present any

4

significant difference in adherence to polystyrene when compared with E. coli K-12

5

DH5α indicating that induction of the SOS system can affect the adherence by E. coli

6

K-12 to abiotic surfaces. Interestingly, despite the nfi mutant did not increase the

7

filamentation it showed an increase in biofilm formation.

8 9

It is known that oxygen influences biofilm formation in E. coli K-12, since

10

oxygen tension is identified as one of the factors that contribute to the dispersion of

11

mature biofilms (Karatan & Watnick, 2009). Nevertheless, some authors have shown

12

that E. coli K-12 strains are able to form biofilms under anaerobic conditions,

13

depending on the conditions of the experiment (Cabellos-Avelar et al., 2006; Bjergbaek

14

et al., 2007).

15 16

In this study, we report on E. coli strains (AB1157, BW9091 and BW535) with

17

the ability to adhere to polystyrene in the absence of oxygen, but at different levels. In

18

aerobiosis, the mutants xthA (BW9091) adhered to the polystyrene substratum like the

19

wild-type strain (AB1157), but in anaerobiosis, a significant decrease was observed in

20

its adherence to polystyrene. The triple mutant BW535 (xthA nfo nth) presented a

21

significant decrease in its adherence to polystyrene when compared with the wild-type

22

strain (AB1157) under both conditions. However we can attest that the reduction in the

23

number of BER mutants adherent to polystyrene under anaerobic conditions was not

24

related to the death of the bacterial strains. In this case, lesions generated in DNA of

19

1

BER strains may lead to filamentation and SOS induction in biofilms. Probably BER

2

participates subsequently removing the terminal left by action of endonuclease V. In

3

this way, the gap formed by endonuclease V are not repaired in the mutants deficient in

4

BER leading to increased frequency of mutations which could reduce biofilm formation.

5 6

Several authors have found that biofilm growth can produce extensive genetic

7

diversity in bacterial populations even though no external stress or mutagen is applied.

8

Diverse genetic variants are generated by biofilms of Pseudomonas aeruginosa,

9

Pseudomonas

fluorescens,

Vibrio

cholerae,

Streptococcus

pneumoniae,

and

10

Staphylococcus aureus, and the formation of bacterial subpopulations interferes with the

11

characteristics of biofilms (Waite et al., 2001; Allegrucci & Sauer, 2007; Deziel et al.,

12

2001; Kirisits et al., 2005; Palmer & Stoodley, 2007; Boles et al., 2004; Yarwood et al.,

13

2007). Boles & Singh, 2008, showed that the presence of double-stranded DNA breaks

14

generated under stress conditions increased the adaptability to environmental changes in

15

biofilms of the pathogen Pseudomonas aeruginosa and that this was due to an increase

16

in the mutation frequencies leading to the formation of genetic variants when these

17

breaks were repaired by a mutagenic mechanism involving recombinatorial DNA repair

18

genes.

19 20

The presence of an extracellular matrix involving bacteria is an important factor

21

for the development of a mature biofilm, and the composition of this matrix is highly

22

complex and influenced by many environmental factors as well as the characteristics of

23

the strains tested (Ghannoum & O'Toole, 2001; Beloin et al., 2008).

24

20

1

The scanning electron micrographs showed that the strains tested (AB1157,

2

BW9091 and BW535) form microcolonies on glass surfaces under aerobic and

3

anaerobic conditions. However, a structure suggestive of an extracellular matrix was

4

observed only in the wild-type strain (AB1157) and the xthA mutant (BW9091) in

5

aerobiosis. Bjergbaek et al., 2007 showed that the oxygen tension affects the

6

morphology of biofilms. Musken et al., observed in 2008 that enterohemorrhagic

7

Escherichia coli (EHEC) expresses a type of fimbriae only under anaerobic conditions.

8

Moreover, as mentioned, the oxygen tension also regulates the expression of an

9

adhesion (Ag43) (Schembri & Klemm, 2001).

10 11

In this paper no extracellular matrix structure was observed in the triple mutant

12

BW535, and this mutant formed less biofilm, both in aerobiosis and in anaerobiosis.

13

Janion et al., 2003 showed that a triple mutant for the xth, nfo and nth genes, deficient in

14

BER, which chronically triggered the SOS system, exhibited a strong filament

15

formation. The fact that this chronically triggered the SOS system may reflect both the

16

quantity and morphology of the biofilm formation for this strain.

17 18

Recently, it has been shown that the induction of the SOS response influences

19

biofilm formation. Gotoh et al., 2010, showed that the mutant of P. aeruginosa, which

20

chronically triggered the SOS system, exhibited a decrease in biofilm formation. Gotoh

21

et al., 2008, showed that different environmental stresses lead to the induction of the

22

SOS system; the induction of this system is responsible for increasing the ability of

23

bacterial strains to form biofilms. In our work we show that the induction of the SOS

24

occurs in biofilms, even without the influence of an external inducing agent.

21

1

Studies have shown that filamentation should not be considered merely the

2

response of the over-stressed, sick, and dying members of the population, but also as a

3

form of survival of the bacterial community. Uropathogenic E. coli (UPEC) employ

4

filamentation as a means to evade immune response. The study by Justice et al., 2006,

5

proposed that components of innate immune response such as oxidative radicals may

6

induce the SOS response and bacterial filamentation.

7 8

All data presented in the present work show the influence of bacterial interaction

9

with the abiotic surface, leading to the induction of the SOS system with the associated

10

filamentation response. Furthermore, we show that BER mutants present more filaments

11

as compared to the isogenic wild strain in the biofilm formed. The relevance of this data

12

points out that responses such as the SOS or are involved in the defense of the genome

13

of planktonic cells against environmental agents and that they can be triggered when

14

microorganisms adhere to abiotic surfaces, which is essential for the colonization of

15

commensal bacteria and/or pathogens. In contrast, nfi mutant does not induce the SOS

16

and filamentation associated in both aerobic or anaerobic conditions, but this mutant

17

was able to form biofilms more intense than the 042 strain used as positive control.

18

Therefore, other pathways beside induction of the SOS could participate in biofilm

19

formation.

20

22

1 2

Acknowledgements

3

This work was supported by a grant from FAPERJ, CNPq, CAPES, SR-2/UERJ and

4

Programa de Núcleo de Excelência (PRONEX) of the Brazilian Ministry of Science and

5

Technology. We thank Bernard Weiss for generously providing the bacterial strains and

6

plasmids.

7 8 9 10 11 12 13 14 15 16 17 18 19

23

1 2

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12 13 14 15 16

31

1

Fig. 1. Filamentous growth on E. coli K12 adherence to glass after 18 h incubation at

2

37ºC, under aerobic condition. (a) Wild type strain (AB1157), adhered to glass with rare

3

filamentous cells; ((b) xthA mutant (BW9091) and (c) triple mutant xthA nfo nth

4

(BW535) presented intense filamentation (arrows);; (d) lexA Ind- mutant (DM49), was

5

negative for filamentation. Magnification 1000 X.”

6 7

Fig 2. Filamentous growth on adherence to glass after 18 h incubation at 37ºC, under

8

anaerobic condition. (a) Wild type strain (AB1157), presented rare filamentous growth;

9

(b) xthA mutant (BW9091) presented intense filamentation (arrows); (c) Triple mutant

10

xthA nfo nth (BW535) presented intense filamentation when adhered to glass (arrows);

11

(d) lexA Ind- mutant (DM49), no exhibited filamentation when adhered to glass.

12

Increase: 1000X.

13 14

Fig. 3. Induction of a sfi::lacZ fusion in E. coli K12 PQ35 after adherence to glass and

15

bacterial strain grown in DMEM in absence and presence of oxygen. *The results are

16

calculated according to the ratio R of b-galactosidase units (B) to alkaline phosphatase

17

units (P) (R = Abs420nmB × incubation time, in min/ Abs420nmP × incubation time, in

18

min) (ap = 0.0400; bp = 0.0043; cp=0.0008 (Glass x DMEM in aerobiosis) ; dp = 0.0488

19

(Glass x DMEM in anaerobiosis)).

20 21

Fig. 4. Quantitative adherence to polystyrene substratum by E. coli K-12 strains in

22

presence and absence of oxygen. ap < 0.05, EAEC 042 × all strains tested in aerobiosis

23

and anaerobiosis; bp < 0.05, DH5α × AB1157, BW9091 and BW535 in aerobiosis and

24

anaerobiosis; *p = 0.0061; **p = 0.0402; ***p = 0.0066.

25

32

1

Fig. 5. Quantitative adherence to polystyrene substratum by E. coli EAEC strains in

2

presence and absence of oxygen. ap < 0.05, BW1466 (wild-type) and BW1506 (nfi)

3

strains tested in aerobiosis and anaerobiosis; *p = 0.0004; **p = 0.0005.

4 5

Fig. 6. Confocal laser scanning micrographs illustrating the viability of E. coli K12

6

strains adhered to glass in aerobiosis (a,c,e) and anaerobiosis (b,d,f), after 18 h of

7

incubation at 37oC, in DMEM. (a) Wild type strain (AB1157) in aerobiose and (b)

8

anaerobiose; (c) xthA mutant (BW9091) in aerobiose and (d) anaerobiose. (e) triple

9

mutant xthA nfo nth (BW535). The mutants xthA (BW9091) and xthA nfo nth (BW535)

10

presented intense filamentation (arrows) in both conditions. Magnification 40 X.”

11 12 13

Fig. 7. Scanning electron micrographs of E. coli K12 strains adhered to glass in

14

aerobiosis (a,c,e) and anaerobiosis (b,d,f), after 18h of incubation at 37oC in DMEM.

15

(a,b) Wild type strain (AB 1157); (c, d) xthA mutant (BW9091) and (e,f) triple mutant

16

xthA nfo nth (BW535), showing intense filamentation (arrows) in both conditions. Bar 5

17

μm.

18 19 20 21 22 23

33

1

Table 1. Bacterial strains derived from E. coli K-12 used in this study Strain

Relevant genotype

Source

AB1157

Wild type

J.D. Hall and P.Howard-Flanders1

BW9091

xthA

B. Weiss2

BW535

nfo-1::kan nth-

B. Weiss2

1::kanΔ(xth-pncA)90 DM49

lexA3

J.D. Hall and P. Howard-Flunders1

PQ35

sfiA::Mud(Ap lac)

P. Quillard and M. Hofnung3

BW1506

cts lacΔU169

B. Weiss2

BW1466

nfi-1:: cat ara Δ

B. Weiss2

(gpt-lac) tnA lac nfi wild type 2

3 4

1

J.D. Hall and P.Howard-Flanders- Univ. of Yale, USA

5

2

B. Weiss- Emory University, Atlanta, GA, USA

6

3

P. Quillardet and M. Hofnung- Institut Pasteur, Paris, France

7 8 9 10 11

34

1

Table 2. Filamentation levels expressed by bacterial strains during interaction with

2

glass Aerobiosis

Anaerobiosis

E. coli K-12 strains

Filamentation *

Filamentation

AB1157 (wild- type)

1.17

0.57

BW9091 (xthA)

2.7

1.33

BW535 (nfo,nth xthA)

4.93

1.87

DM49 (lexAInd-)

0

0

BW1466 (nfi wild-type)

0.6

0.05

BW1506 (nfi)

0

0

3 4 5

*The intensity of filamentation, for each strain, was correspondent to the number of

6

filaments observed in 30 microscope fields.

7 8 9 10

35

β - Galactosidase units/ alkaline phosphatase units *

b

15

Aerobiosis Anaerobiosis

c 10

a d

5

0 DMEM-grown cells glass-attached cells

E. coli K-12 PQ35

1.0

a

** *

0.4

b 0.2

E. coli strains

5α D H

04 2

49 D M

W 53 5 B

B 11 57 B W 90 91

0.0

A

OD570nm

0.8

0.6

Aerobiosis Anaerobiosis

***

A na er o bi os is

A er ob io si s

OD 570nm

* **

3

1466 1506

2

1

0