![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Applied and Environmental Microbiology, September 1999, p. 4285-4287, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
andDepartment of Biology, Southwest Texas State University, San Marcos, Texas 78666-4616
Received 24 March 1999/Accepted 18 June 1999
 |
ABSTRACT |
|---|
Top
Abstract
Text
References
|
|---|
Slow growth has been hypothesized to be an essential aspect of
bacterial physiology within biofilms. In order to test this hypothesis,
we employed two strains of Escherichia coli, ZK126 (
lacZ rpoS+) and its isogenic
rpoS derivative, ZK1000. These strains were grown at two
rates (0.033 and 0.0083 h
1) in a glucose-limited
chemostat which was coupled either to a modified Robbins device
containing plugs of silicone rubber urinary catheter material or to a
glass flow cell. The presence or absence of rpoS did not
significantly affect planktonic growth of E. coli. In
contrast, biofilm cell density in the rpoS mutant strain
(ZK1000), as measured by determining the number of CFU per square
centimeter, was reduced by 50% (P < 0.05). Deletion
of rpoS caused differences in biofilm cell arrangement, as
seen by scanning confocal laser microscopy. In reporter gene
experiments, similar levels of rpoS expression were seen in
chemostat-grown planktonic and biofilm populations at a growth rate of
0.033 h1. Overall, these studies suggest that
rpoS is important for biofilm physiology.
| |
TEXT |
|---|
|
Top
Abstract
Text
References
|
|---|
In their natural environments, bacteria often adhere to surfaces on which they form biofilm communities that may be several millimeters thick. Within biofilms, individual bacteria are encased in a polysaccharide matrix, which functions to bind cells together and facilitates adhesion to the underlying surface. Bacteria are not distributed uniformly throughout a biofilm but rather aggregate into microcolonies, which are typically a few micrometers in diameter (6). Studies employing scanning confocal laser microscopy (SCLM) have shown a wide range of bacterial growth rates throughout a biofilm. The fastest growth was observed at the biofilm-liquid interface. Bacteria in the biofilm interior, particularly those inside microcolonies, grew much more slowly, presumably due to limited access to nutrients (10, 13). These and other studies have led to the hypothesis that slow growth is a major aspect of bacterial biofilm physiology (4). In order to test this hypothesis, we investigated whether the absence of a slow-growth-activated gene, rpoS (7), could affect the biofilm formation of Escherichia coli under defined growth conditions (15). Here we report that deletion of rpoS greatly reduces the ability of E. coli to grow in biofilms yet has little effect on the growth of planktonic (i.e., unattached) bacteria.
(This research was conducted by J. L. Adams in partial fulfillment of the requirements for an M.S. from Southwest Texas State University.)
Strains and culturing conditions.
The strains of E. coli used in this study are ZK126 (lacZ), ZK1000
(ZK126 rpoS) (1), and DS526 (ZK126 
RZ5
rpoS742::lacZ) (13a).
Cultures were stored frozen at 
80°C in Luria-Bertani (LB) broth
containing 15% (vol/vol) glycerol as described elsewhere (14). Prior to each experiment, the appropriate E. coli strain was streaked from a frozen stock culture onto LB agar,
checked for purity, and grown overnight in 5 ml of glucose-limited,
defined medium (GDM) (15) containing 0.25 g of glucose
per liter.
Biofilm chemostat experiments.
Chemostats were coupled to a
modified Robbins device (MRD; Tyler Research, Edmonton, Alberta,
Canada) as described by Whiteley et al. (15). Briefly, this
consisted of filling a chemostat with sterile GDM and inoculating it
with 1 ml of an overnight E. coli culture in GDM. This
culture was allowed to grow overnight under batch conditions, after
which continuous culture was commenced at a dilution rate (DR) of
either 0.033 or 0.0083 h1. The chemostat cultures were
allowed to equilibrate for 1 generation time (121 h at a DR of 0.0083 h1 and 30 h at a DR of 0.033 h1),
after which time the chemostat was connected to an MRD containing 7-mm-diameter silicone rubber plugs. A peristaltic pump was used to
circulate the chemostat culture through the MRD at a flow rate of 100 ml min1. After 48 h, the experiment was stopped and
nine plugs were removed from the MRD, sonicated, serially diluted in
phosphate-buffered saline, and plated onto LB agar (Difco Laboratories,
Detroit, Mich.) as previously described (8, 15). Each
chemostat-MRD culture experiment was replicated a minimum of three
times. Within each chemostat-MRD replicate, a minimum of five
measurements were taken.
1. For SCLM examination, the glass slide was
removed and stained with BacLite Live/Dead viability stain (Molecular
Probes, Eugene, Oreg.) in order to estimate the viability of individual
cells. Biofilm formation in flow cells was examined by SCLM with an
Olympus IX-70 inverted microscope (Olympus America Inc., Melville,
N.Y.) coupled with a Bio-Rad 1024 SCLM System (Bio-Rad Laboratories, Hercules, Calif.). The slides were placed with the biofilm side facing
the 60× Uplan Apo (Olympus) oil immersion objective lens.
rpoS expression assay.
In order to compare the
levels of rpoS expression in biofilm cells and planktonic
cells, reporter strain DS526, containing an
rpoS::lacZ fusion on a phage, was
constructed by D.A. Siegele, Texas A&M University, as previously
described (5). This strain was cultured in the chemostat-MRD
apparatus at a DR of 0.033 h1 as described above. After
48 h of biofilm growth, biofilm and planktonic samples were
removed and frozen at 80°C for 2 weeks until analyzed. We
permeabilized E. coli cells with chloroform and sodium
dodecyl sulfate and quantified the 
-galactosidase activity with
o-nitrophenyl--D-galactopyranoside (ONPG) as
described by Miller (9). The cell number was determined on
the basis of direct cell counts of 4',6-diamidino-2-phenylindole
(DAPI)-stained biofilm and planktonic cell suspensions.
-Galactosidase activity was expressed as nanomoles of ONPG cleaved
per cell per minute.
Data analysis. Biofilm cell densities, expressed as log10 CFU per square centimeter, and planktonic cell densities, expressed as log10 CFU per milliliter, were analyzed by one-way analysis of variance.
The influences of rpoS deletion and growth rate on E. coli biofilms and planktonic cultures are shown in Fig. 1. As can be seen, deletion of rpoS had a major impact on biofilm populations and less of an impact on planktonic populations. SCLM examinations (Fig. 2) showed differences in E. coli biofilm structures in the presence (Fig. 2A) and absence (Fig. 2B) of rpoS. When bacteria were grown at a DR of 0.033 h1, similar levels of -galactosidase activity were
seen in biofilm (3.04 × 106 nmol of ONPG
min1 cell1) and planktonic (3.08 × 106 nmol of ONPG min1 cell1)
populations of E. coli DS526 containing an
rpoS::lacZ fusion.
|
|
1. In this experiment, rpoS
expression, as indicated by the amount of -galactosidase activity
per cell, was equivalent in both planktonic (3.08 × 106 nmol of ONPG min1 cell1)
and biofilm (3.04 × 106 nmol of ONPG
min1 cell1) populations. Our observations
are consistent with those of Notley and Ferenci (12), who
observed rpoS expression in chemostat cultures of E. coli at a DR of 
0.2 h1. Deletion of
rpoS had an impact on biofilm cell density (Fig. 1) and cell
arrangement (Fig. 2), so it is likely that rpoS expression is more important to biofilm populations than to planktonic populations.
| |
ACKNOWLEDGMENTS |
|---|
This project was supported in part by a grant from the Advanced Research Program of the Texas Higher Education Coordinating Board and by the Biology Department at Southwest Texas State University. The scanning confocal microscope used in this study was purchased with funds from an NSF-ILI grant.
We thank Debby Siegele, Texas A&M University, and Grant Balzer, Joe Koke, and Jim Ott, Southwest Texas State University, for their help and suggestions.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Biology, Southwest Texas State University, 601 University Dr., San Marcos, TX 78666-4616. Phone: (512) 245-3365. Fax: (512) 245-8713. E-mail: RM12{at}swt.edu.
Present address: Dynamac Corporation, Kennedy Space Center, FL 32899.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Bohannon, D. E.,
N. Connell,
J. Keener,
A. Tormo,
M. Espinosa-Urgel,
M. M. Zambrano, and R. Kolter.
1991.
Stationary-phase-inducible "gearbox" promoters: differential effects of katF mutations and role of  70.
J. Bacteriol.
173:4482-4492 |
| 2. |
Davies, D. G.,
M. R. Parsek,
J. P. Pearson,
B. H. Iglewski,
J. W. Costerton, and E. P. Greenberg.
1998.
The involvement of cell-to-cell signals in the development of a bacterial biofilm.
Science
280:295-298 |
| 3. |
Evans, D. J.,
M. R. W. Brown,
D. G. Allison, and P. Gilbert.
1990.
Susceptibility of bacterial biofilms to tobramycin: role of specific growth rate and phase in the division cycle.
J. Antimicrob. Chemother.
25:585-591 |
| 4. | Gilbert, P., J. Das, and I. Foley. 1997. Biofilm susceptibility to antimicrobials. Adv. Dent. Res. 11:160-167[Abstract]. |
| 5. |
Lange, R., and R. Hengge-Aronis.
1994.
The cellular concentration of the sigma-S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation and protein stability.
Genes Dev.
8:1600-1612 |
| 6. |
Lawrence, J. R.,
D. R. Korber,
B. D. Hoyle,
J. W. Costerton, and D. E. Caldwell.
1991.
Optical sectioning of microbial biofilms.
J. Bacteriol.
173:6558-6567 |
| 7. | Loewen, P. C., B. Hu, J. Strutinsky, and R. Sparling. 1998. Regulation in the rpoS regulon of Escherichia coli. Can. J. Microbiol. 44:707-717[Medline]. |
| 8. | McLean, R. J. C., M. Whiteley, B. C. Hoskins, P. D. Majors, and M. M. Sharma. Laboratory techniques for studying biofilm growth, physiology, and gene expression in flowing systems and porous media. Methods Enzymol., in press. |
| 9. | Miller, J. H. 1972. Experiments in molecular genetics, p. 352-355. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 10. | Møller, S., A. R. Pedersen, L. K. Poulsen, E. Arvin, and S. Molin. 1996. Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy. Appl. Environ. Microbiol. 62:4632-4640[Abstract]. |
| 11. |
Nickel, J. C.,
I. Ruseska,
J. B. Wright, and J. W. Costerton.
1985.
Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material.
Antimicrob. Agents Chemother.
27:619-624 |
| 12. |
Notley, L., and T. Ferenci.
1996.
Induction of RpoS-dependent functions in glucose-limited continuous culture: what level of nutrient limitation induces the stationary phase of Escherichia coli?
J. Bacteriol.
178:1465-1468 |
| 13. |
Poulsen, L. K.,
G. Ballard, and D. A. Stahl.
1993.
Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms.
Appl. Environ. Microbiol.
59:1354-1360 |
| 13a. | Siegele, D. A. Unpublished data. |
| 14. |
Siegele, D. A., and J. C. Hu.
1997.
Gene expression from ParaBAD plasmids at subsaturating inducer concentrations represents mixed populations.
Proc. Natl. Acad. Sci. USA
94:8168-8172 |
| 15. | Whiteley, M., E. Brown, and R. J. C. McLean. 1997. An inexpensive chemostat apparatus for the study of microbial biofilms. J. Microbiol. Methods 30:125-132. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | Microbiol. Mol. Biol. Rev. | Eukaryot. Cell | All ASM Journals |
|---|
