MiR-34a inhibits lymphatic metastasis potential of mouse hepatoma cells

Genetic adaptation is one of the key features of Escherichia coli (E. coli) that ensure its survival in different hostile environments. E. coli seems to initiate biofilm development in response to specific environmental cues. A number of properties inherent within bacterial biofilms indicate that their gene expression is different from that of planktonic bacteria. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to a varied number of stresses, as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stress environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. The purpose of this study was to understand and analyse the responses of rpoS and bolA gene to sudden change in the environment. In this study, E. coli K-12 MG1655, rpoS, and bolA mutant strains were used and gene expression was studied. Results show that both genes contribute to the ability to respond and adapt in response to various types of stresses. RpoS response to various stress environments was somehow constant in both the planktonic and biofilm phases, whereas bolA responded well under various stress conditions, in both planktonic and biofilm mode, up to 5-6-fold change in the expression was noticed in the case of pH variation and hydrogen peroxide stress (H(2)O(2)) as compared with rpoS.     

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).     

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.     

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.     

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.     

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.     

Interrelationships between colonies, biofilms, and planktonic cells of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic human pathogen that causes chronic infections in immunocompromised individuals. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms. The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems. Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase. However, few, if any, studies have examined global gene expression in colonies. We used a proteomic approach to investigate the interrelationships between planktonic cells, colonies, and biofilms under comparable conditions. Our results show that protein profiles in colonies resemble those of planktonic cells. Furthermore, contrary to what has been reported previously, the protein profiles of biofilms were found to more closely resemble those of exponentially growing planktonic cells than those of planktonic cells in the stationary phase. These findings raise some intriguing questions about the true nature of biofilms.     

Chemical composition of Enterococcus faecalis in biofilm cells initiated from different physiologic states

Enterococcus faecalis is a ubiquitous bacterium of the gut that is observed in persistent periradicular infections. Its pathogenicity is associated with biofilm formation and the ability to survive under nutrient-poor (starvation) conditions. However, characteristics of chemical composition of biofilm cells developed by starved E. faecalis cells remain poorly understood. In this study, E. faecalis cells in exponential, stationary, and starvation phases were prepared and separately cultured to form biofilms. Confocal laser scanning microscopy was performed to verify biofilm formation. Raman microscopy was used to investigate the chemical composition of cells within the biofilms. Compared to cells in exponential or stationary phase, starved cells developed biofilms with fewer culturable cells (P?E. faecalis.     

A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode

Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (D-ribose-binding periplasmic protein, D-galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode.     

The Small Molecule DAM Inhibitor,Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro

Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam) alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC). In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell-clumps were scattered and attached to the bottom of the plate when cells were grown in the presence of pyrimidinedione. Scanning electron microscopy analysis demonstrated the absence of an extracellular polysaccharide matrix in pyrimidinedione-grown biofilms compared to control-biofilms. Pyrimidinedione also significantly inhibited MRSA, MSSA, and Staphylococcus epidermidis biofilm growth in vitro. Furthermore, pyrimidinedione does not exhibit eukaryotic cell toxicity. In a microarray analysis, 56 genes were significantly up-regulated and 204 genes were significantly down-regulated. Genes involved in galactose metabolism were exclusively up-regulated in pyrimidinedione-grown biofilms. Genes related to DNA replication, cell division and the cell cycle, pathogenesis, phosphate-specific transport, signal transduction, fatty acid biosynthesis, protein folding, homeostasis, competence, and biofilm formation were down regulated in pyrimidinedione-grown biofilms. This study demonstrated that the small molecule Dam inhibitor, pyrimidinedione, inhibits pneumococcal biofilm growth in vitro at concentrations that do not inhibit planktonic cell growth and down regulates important metabolic-, virulence-, competence-, and biofilm-related genes. The identification of a small molecule (pyrimidinedione) with S. pneumoniae biofilm-inhibiting capabilities has potential for the development of new compounds that prevent biofilm formation.     

An expanded regulatory network temporally controls Candida albicans biofilm formation

Candida albicans biofilms are composed of highly adherent and densely arranged cells with properties distinct from those of free‐floating (planktonic) cells. These biofilms are a significant medical problem because they commonly form on implanted medical devices, are drug resistant and are difficult to remove. C. albicans biofilms are not static structures; rather they are dynamic and develop over time. Here we characterize gene expression in biofilms during their development, and by comparing them to multiple planktonic reference states, we identify patterns of gene expression relevant to biofilm formation. In particular, we document time‐dependent changes in genes involved in adhesion and metabolism, both of which are at the core of biofilm development. Additionally, we identify three new regulators of biofilm formation, Flo8, Gal4, and Rfx2, which play distinct roles during biofilm development over time. Flo8 is required for biofilm formation at all time points, and Gal4 and Rfx2 are needed for proper biofilm formation at intermediate time points.     

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.