Significance of rpoS during maturation of Escherichia coli biofilms

Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.     

Impact of rpoS deletion on the proteome of Escherichia coli grown planktonically and as biofilm

To investigate the role of rpoS in gene expression of Escherichia coli cells grown as biofilms, we compared the proteomes of a rpoS mutant and the wild-type strain. Experiments were performed on planktonic cells (in exponential or stationary growth phase) and biofilms developed on glass wool. Spot-by-spot comparison of gels obtained from biofilm and planktonic wild-type organisms showed that the intensity of between 22 and 30% of detected spots was affected by the growth mode, depending of the control used. Principal component analysis, used to interpret the variations in protein spot densities, discriminated exponential-phase cells (wild-type and mutant) from the other incubation conditions and secondarily 72-old cultures. The statistical analysis demonstrated that the rpoS mutation did not significantly modify the proteome of exponential-growth phase cells, the differences involving only 3% of the proteome. However, increasing the incubation time from 8 to 72 h noticeably increased the number of changed proteins. A cluster analysis showed that RpoS plays a role in the special nature of the gene expression of biofilm cells but lower than in stationary-phase bacteria. We identified 35 rpoS-regulated proteins that were already or not described as controlled by this sigma factor. For some of them, the mode of regulation by RpoS was obviously dependent on the culture condition (planktonic vs biofilm).     

Contribution of rpoS and bolA genes in biofilm formation in Escherichia coli K-12 MG1655

Flexibility of gene expression in bacteria permits its survival in varied environments. The genetic adaptation of bacteria through systematized gene expression is not only important, but also clinically relevant in their ability to grow biofilms in stress environments. Stress responses enable their survival under more severe conditions, enhanced resistance and/or virulence. In Escherichia coli (E. coli), two of the possible important genes for biofilm growth are rpoS and bolA gene. RpoS is also called as a master regulator of general stress response. Even though many studies have revealed the importance of rpoS in planktonic cells, little is known about the functions of rpoS in biofilms. In contrast, bolA which is a morphogene in E. coli is overexpressed under stressed environments resulting in round morphology. The hypothesis is that bolA could be implicated in biofilm development. This study reviewed the literature with the aim of understanding the stress tolerance response of E. coli in relation with rpoS and bolA genes in different environmental conditions including heat shock, cold shock, and stress in response to oxidation, acidic condition and in presence of cadmium. Knowledge of the genetic regulation of biofilm formation may lead to the understanding of the factors that drive the bacteria to switch to the biofilm mode of growth.     

Identification and characterization of a high-affinity zinc uptake system in Neisseria gonorrhoeae

Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic culture. RpoS levels in biofilms, indicated by immunoblot analysis, were similar to levels in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.     

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.     

Visible laser and UV-A radiation impact on a PNP degrading Moraxella strain and its rpoS mutant

The role of stationary phase sigma factor gene (rpoS) in the stress response of Moraxella strain when exposed to radiation was determined by comparing the stress responses of the wild-type (WT) and its rpoS knockout (KO) mutant. The rpoS was turned on by starving the WT cultures for 24 h in minimal salt medium. Under non-starved condition, both WT and KO planktonic Moraxella cells showed an increase in mortality with the increase in duration of irradiation. In the planktonic non-starved Moraxella, for the power intensity tested, UV radiation caused a substantially higher mortality rate than did by the visible laser light (the mortality rate observed for 15-min laser radiation was 53.4 +/- 10.5 and 48.7 +/- 8.9 for WT and KO, respectively, and 97.6 +/- 0 and 98.5 +/- 0 for 25 s of UV irradiation in WT and KO, respectively). However, the mortality rate decreased significantly in the starved WT when exposed to these two radiations. In comparison, rpoS protected the WT against the visible laser light more effectively than it did for the UV radiation. The WT and KO strains of Moraxella formed distinctly different types of biofilms on stainless steel coupons. The KO strain formed a denser biofilm than did the WT. Visible laser light removed biofilms from the surfaces more effectively than did the UV. This was true when comparing the mortality of bacteria in the biofilms as well. The inability of UV radiation to penetrate biofilms due to greater rates of surface absorption is considered to be the major reason for the weaker removal of biofilms in comparison to that of the visible laser light. This result suggests that high power visible laser light might be an effective tool for the removal of biofilms.     

Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of Escherichia coli

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA'-'lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.     

Effect of Escherichia coli morphogene bolA on biofilms

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.     

Enterobactin is required for biofilm development in reduced-genome Escherichia coli

A variety of bacterial cell surface structures and quorum signalling molecules play a role in biofilm development in Escherichia coli. However, here we show that an engineered reduced-genome E. coli mutant that lacks 17.6% of the parental E. coli genome, including the genes involved in the synthesis of various cell surface structures, such as type 1 fimbriae, curli, exopolysaccharide polymers and the autoinducer-2 signalling molecule, is able to develop mature biofilms. Using temporal gene expression profiling, we investigated phenotypic changes in reduced-genome biofilms in relation with the genes encoding the synthesis of different amino acids that were differentially expressed during biofilm formation. We identified and characterized entB, marR, dosC, mcbR and yahK genes, as involved in biofilm formation by the reduced-genome E. coli. Of these, for a first time, we demonstrated that overproduction of entB and yahK, which encode an enterobactin for iron transport and a hypothetical oxidoreductase protein, respectively, promoted biofilm development and maturation. Our results indicate that specific types of genes contribute to phenotypic changes in reduced-genome E. coli biofilms. In addition, this work demonstrates that the functions of biofilm-specific genes could be analysed through experiments using the reduced-genome E. coli.     

Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen) and surE (stationary-phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis SurE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in co-ordinating development of the stress resistance for passage from the present to the next host cells through its regulon.     

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of a large number of genes in Escherichia coli under stress conditions. The present study investigated the growth characteristics and metabolic pathways of rpoS gene knockout mutant of E. coli growing in LB media under aerobic condition. The analyses were made based on gene expressions obtained by DNA microarray and RT-PCR, enzyme activities and intracellular metabolite concentrations at the exponential and early stationary phases of growth. Although the glucose utilization pattern of the mutant was similar to the parent strain, the mutant failed to utilize acetate throughout the cultivation period. Microarray data indicated that the expression levels of several important genes of acetate metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in the absence of rpoS gene. Interestingly, there was an increased activity of TCA cycle during the exponential growth phase, which was gradually diminished at the onset of stationary phase. Moreover, rpoS mutation had profound effect on the expression of several other genes of E. coli metabolic pathways that were not described earlier. The changes in the gene expressions, enzyme activities and intracellular metabolite concentrations of the rpoS mutant are discussed in details with reference to the major metabolic pathways of E. coli.