Applied and Environmental Microbiology, September 2004, p . 5682-5684, Vol . 70, No . 9

Effect of Escherichia coli Morphogene bolA on Biofilms

Helena L . A . Vieira, Patrick Freire, and Cecília M . Arraiano^*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal

Received 28 January 2004/ Accepted 28 April 2004

ABSTRACT

Biofilm physiology is established under a low growth rate . The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development . In order to verify this hypothesis, we tested the effect of bolA on biofilm formation . Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms .

In natural environments, most bacteria live attached to surfaces in structures known as biofilms, rather than existing as isolated planktonic cells (14) . Biofilm formation is such a common phenomenon that almost any material coming into contact with bacteria present in fluids, e.g., blood or seawater, can become covered with biofilms . Given the important medical and economic consequences of this phenomenon, understanding the colonization process would help in the design of methods to prevent biofilm formation (9) . Several properties inherent within bacterial biofilms indicate that gene expression in biofilm-associated bacteria is different from that observed in planktonic bacteria (13) . Biofilm-associated cells demonstrate increased resistance to many toxic substances, such as antibiotics, detergents, and host immune defense response products . Furthermore, bacteria within biofilms encounter high-osmolarity conditions, oxygen limitations, and high cell density (9) . Slow growth is also an important aspect of bacterial biofilm physiology (1, 10) . Altogether, these results suggest that biofilm formation is a response to unfavorable environments, since biofilms are better adapted to different types of stress than are planktonic bacteria .

Stress response genes are induced whenever a cell needs to adapt and survive under adverse growth conditions . The Escherichia coli morphogene bolA is an example of those genes . It causes round morphology when overexpressed (2) . bolA was first described as a gene involved in adaptation to stationary-phase growth under the control of a sigma factor (^S), and bolA mRNA levels are inversely proportional to growth rate (3, 4, 6) . However, the function of bolA is not confined to stationary phase; bolA expression is induced by several forms of stress during early growth phase, such as heat shock, acidic stress, oxidative stress, and sudden carbon starvation (11) .

As bolA expression and biofilm formation are two events related to stationary phase and stress, we tested the hypothesis that bolA could be implicated in biofilm development .

The strains used were E . coli MC1061 [F^– araD139 (ara-leu)7697 (lac)X74 galU galK strA] (3), SK6582 [MC1061 bolA2::Kan] (3), and CMA14 [MC1061/pMAK580] (12) . Plasmid pMAK580 (2) contains bolA under regulation of its own promoters . During the experiments, batch cultures were launched from overnight growths (performed with Luria broth medium) that were diluted to an optical density measured at 620 nm of 0.08 . Cultures were grown aerobically in M9 minimal medium (7) containing 0.4% (wt/vol) glucose (Merck) at 37°C and with shaking at 120 rpm on an orbital shaker . The medium was supplemented as required with 0.8 mM leucine, 20 mg of chloramphenicol ml^–1, 50 mg of kanamycin ml^–1, and 25 mg of streptomycin ml^–1 (all from Sigma) .

The determination of biofilm thickness in microtiter plates was carried out as described by O'Toole and Kolter (8) . Briefly, cells were allowed to grow in M9 minimal medium for 24 h in 96-well polystyrene microtiter dishes for stationary-phase-growth analysis . Unattached bacteria existing in the culture medium were removed, and the biofilm was stained with 0.2% (wt/vol) crystal violet for 15 min (this dye stains the cells but not the polystyrene) . The excess crystal violet dye was washed out, and the samples were washed three times with bidistilled water . Ethanol was added to the wells to release the dye, and the optical density at 600 nm was measured in order to estimate the amount of biofilm formed (8) . The results showed that bolA overexpression induced biofilm development, since the optical density of crystal violet-stained biofilms was about fourfold higher for the culture of the strain overexpressing bolA than for the culture of the wild type (MC1061) (Fig . 1) . The deletion of bolA decreased biofilm formation (Fig . 1) . The same experiments were done with 0.6% (wt/vol) glucose and 0.8% (wt/vol) glucose (data not shown), and the effect of bolA on biofilm formation at these glucose concentrations was similar to that obtained with 0.4% (wt/vol) glucose . When the three strains were grown in Luria broth, however, there was practically no biofilm formation . This result was expected, since biofilm formation is a response to unfavorable conditions .

 
The same results were confirmed by phase-contrast microscopy (Fig . 2) . Cells were cultured in a six-well polystyrene microtiter dish at 37°C and with shaking at 120 rpm for 48 h . The supernatant was drained, and the sample was washed repeatedly with bidistilled water in order to eliminate planktonic bacteria . Finally, biofilm development was observed directly on the polystyrene with a phase-contrast microscope (Leica DMRB) and a 100x oil objective with a 1.3 aperture . Only in the case of the strain overexpressing bolA (CMA14) were biofilm populations observed . After 16 h, microcolonies could already be observed with a microscope in the CMA14 culture (data not shown) . However, after 48 h in these growth conditions, neither the wild type (MC1061) nor SK6582 (bolA) presented any biofilm structures (Fig . 2) .

 
In order to test biofilm formation under stress conditions, stresses were imposed by following the experimental conditions described by Santos et al . (11) . For carbon limitation, batch cultures were grown in M9 minimal medium supplemented with 0.4% (wt/vol) glucose until they reached an optical density at 620 nm of 0.3, corresponding to exponential phase . At that moment, the stress was imposed, and the cells were transferred to 96-well polystyrene microtiter dishes for a 48-h culture . Sudden depletion of glucose was achieved by harvesting the cells by centrifugation for 10 min at 7,520 x g at 4°C and washing them twice with 2 volumes of sterile ice-cold M9 medium with no carbon source . The cells were resuspended in the same volume of minimal medium with 0.2% (wt/vol) glucose (corresponding to half the normal level of carbon source) . For oxidative stress, H₂O₂ was added to the culture at a final concentration of 15 mM . Under these stress conditions, bolA expression induced biofilm formation (Fig . 3) . In the case of nutrient limitation (0.2% [wt/vol] glucose) and oxidative stress, the presence of bolA in the wild-type strain increased biofilm thickness (measured by crystal violet staining) about fivefold over that induced by the bolA strain . As has been shown previously (11), bolA mRNA levels increase fivefold in stationary phase, but under stress conditions these levels can rise 20-fold, which would explain why the wild-type strain presented greater amounts of biofilm under stress conditions, even in the absence of pMAK580 .

 
Overall, these results suggest a new phenotype for the bolA gene . In addition to its ability to produce a round morphology, bolA is involved in biofilm development . The fact that bolA is expressed under unfavorable conditions (i.e., stress and stationary phase) suggests that biofilm formation is a mode of action by which the bacteria protect themselves against the environment . The expression of bolA is under the transcriptional control of ^S (encoded by rpoS) . The presence or absence of ^S has an impact on biofilms (5) . In rpoS deletion strains, the biofilm cell density is reduced by 50%, and there are also differences in biofilm structure (1) . Curiously, deletion of bolA also reduces biofilm cell density by 50% (Fig . 1) . Taking into account that the levels of bolA depend on ^S, we can speculate that ^S may act via bolA in order to facilitate biofilm development . However, the results do not exclude the possibility that other factors regulated by ^S might also be involved in biofilm formation .

ACKNOWLEDGMENTS

This work has been supported by grants from Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) . P.F . is the recipient of a doctoral fellowship from the Fundação para a Ciência e a Tecnologia .

REFERENCES

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