MicrobiologyBytes

A biofilm is a surface-associated population of microbes that is embedded in a cement of extracellular compounds. This cement is known as matrix. The two main functions of matrix are to protect cells from their surrounding environment, preventing drugs and other stresses from penetrating the biofilm, and to maintain the architectural stability of the biofilm, acting as a glue to hold the cells together. The presence of matrix is a contributing factor to the high degree of resistance to antimicrobial drugs observed in biofilms. Because biofilms have a major impact on human health, and because matrix is such a pivotal component of biofilms, it is important to understand how the production of matrix is regulated.

A group of researchers has identified a novel regulatory gene network that plays an important role in the spread of common, and sometimes deadly, fungus infections. The new findings establish the role of Zap1 protein in the activation of genes that regulate the synthesis of biofilm matrix. Candida albicans is a fungus, more specifically a yeast, which approximately 80 percent of people have in their gastrointestinal and genitourinary tract with no ill effects. However, at elevated levels it can cause non-life threatening conditions like thrush and yeast infections. C. albicans infection becomes much more serious, and can be lethal in those with compromised immune systems who have an implantable medical device such as a pacemaker or artificial joint, or who use broad-spectrum antibiotics. Central to such infections is the biofilm –  a population of microbes, in this case C. albicans cells, joined together to form a sheet of cells. The cells in the biofilm produce extracellular components such as proteins and sugars, which form a cement-like matrix. This matrix serves to protect the cells of the biofilm, preventing drugs and other stressors from attacking the cells while acting as a glue that holds the cells together. By doing this, the matrix provides an environment in which yeast cells in the biofilm can thrive, promoting infection and drug resistance.

Biofilms have a major impact on human health and matrix is such a pivotal component of biofilms. It is important to understand how the production of matrix is regulated. In the study, the scientists found that the zinc-responsive regulatory protein Zap1 prevents the production of soluble beta-1,3 glucan, a sugar that is a major component of matrix. They also identified other genes whose expression is controlled by Zap1, called Zap1 target genes. They found that these genes encode two types of enzymes, glucoamylases and alcohol dehydrogenases, which both govern the production and maturation of matrix components. Understanding this novel regulatory gene network gives us insight into the metabolic processes that contribute to biofilm formation, and the role the network plays in infection. By better understanding the mechanisms by which biofilms develop and grow, we can start to look at targets for combating infection.

Biofilm Matrix Regulation by Candida albicans Zap1. 2009 PLoS Biol 7(6): e1000133 doi:10.1371/journal.pbio.1000133
A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble b-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble b-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection.

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• Candida albicans: the video
• Candida parapsilosis secreted lipase is a major virulence factor
• Bugs Against Biofilms

A recent paper in Trends in Microbiology examined the possibilities for using bacteriophages in controlling biofilms which might result in healthcare-associated infections (Preventing biofilms of clinically relevant organisms using bacteriophage. 2009 Trends in Microbiology 17: 66-72). Biofilms are firmly attached microbial communities in which the organisms produce an extracellular polymeric matrix. Biofilm organisms might cause disease by detachment of individual cells or clumps of cells, by production of endotoxin, or by providing a niche for the development of antibiotic-resistant organisms. Biofilm organisms are usually tolerant to antimicrobial agents and the treatment of indwelling medical device-associated infections with systemic antimicrobial agents is usually ineffective.

Bacteriophages have been used for the treatment of infectious diseases in plants and animals, although few clinical trials with stringent negative controls have been carried out. There is a renewed interest in phage therapy in light of growing concerns with antimicrobial resistance in healthcare institutions worldwide. The use of phages for the treatment of device-associated infections could reduce the use of antibiotics and might limit the spread of resistant organisms.

There is some evidence for the potential of phages in biofilm control. Bacteriophage T4 phage was effective against E. coli biofilms in a glucose-limited chemostat, although the rate of phage synthesis and assembly were directly proportional to the amount of protein synthesis in the host cell. Some phages produce polysaccharide depolymerases that have the potential to degrade the biofilm matrix.

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However, there are several important characteristics of phage that should be considered when evaluating the potential of phages to control clinically relevant biofilms. The specificity of receptors for a single phage strain will determine its host range; some phage have specificity at the strain level, such as strain typing phages, whereas some are more broad-spectrum and can infect multiple strains or related species. This high degree of specificity could be a drawback, especially in the case of polymicrobial biofilms.

Few studies have explored the role of biofilms in the development of phage-resistance. Phage cocktails developed via the isolation of host range mutants and broad spectrum phage could be advantageous. Another important question is how a patient’s immune system will respond to the therapeutic introduction of phage. Phages are antigenic and elicit a response by serum antibodies and the cellular immune system. Repeated exposure to the phage results in increasing antibody titers and studies in animal models have shown that phage is cleared from the bloodstream by the cellular immune system. In a short-lived treatment, the antibody response to phage is weak except in cases were serum antibody titers are present before phage treatment. It is possible, although unproven, that phage might associate with biofilms and thereby be protected from inactivation. It might also be possible to design mutant phages with enhanced ability to resist clearance by the cellular immune system.

Related:

• Biofilms
• Saturday Cimema: Bacteriophage Therapy
• Bacteriophages for plant disease control
• Coevolution with viruses drives bacterial mutations

Bioremediation can be defined as a process that uses microorganisms, fungi, green plants or their enzymes to return the natural environment altered by contaminants to its original condition. A major advantage of bioremediation is its reduced cost compared to conventional cleanup techniques – the cost of remediation for all contaminated sites in the USA alone is estimated to be $1.7 trillion (Molecular approaches in bioremediation. Curr Opin Biotechnol. Nov 12 2008). In addition, bioremediation is often a permanent solution providing complete transformation of the pollutant to its molecular constituents like carbon dioxide and water rather than a partial method that transfers wastes from one phase to another. Unfortunately, there are many man-made compounds that lack good biological catalysts, and many instances where good biocatalysts fail to transform pollutants in the environment.

Bacteria have enormous potential for cleaning up wastes; however, the interactions between bacteria and pollutants are complex and suitable outcomes do not always take place. Hence, molecular approaches are being applied to enhance bioremediation. One advance in bioremediation to improve the stability of the biocatalyst is to create a system where degradation occurs in the area near the roots of plants known as the rhizosphere. In rhizoremediation, the bacteria degrade the pollutants while the plant roots provide a niche for the microorganism and key nutrients. The advantages of rhizoremediation include the ability of plant roots to provide a large surface area for bacterial propagation and biofilm formation, the roots transport the bacteria through the contaminated soil, the roots provide a niche for the bacteria by providing nutrients, and the roots facilitate oxygen exchange. Successful rhizoremediation systems have been established for pollutants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls (PCBs), fuels, metals, and pesticides such as parathion.

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Directed evolution or DNA shuffling is a powerful mutagenesis technique that mimics the natural molecular evolution of genes in order to efficiently re-design them. Its power lies in the fact that it can introduce multiple mutations into a gene in order to create new enzymatic activity, which can be discovered by a suitable method of selection (bioassays). Family shuffling applies DNA shuffling to groups of related genes to combine them in a manner that accelerates directed evolution. Genome shuffling recombines the chromosomes of several bacteria to enhance the activity of the whole organism.

Metabolic engineering involves redirecting a cell’s metabolism to achieve a particular goal using recombinant engineering. This technique has been used to create bacterial strains that degrade chlorinated ethenes through the addition of several cloned enzymes to the cell. Metabolic engineering has also been used successfully to handle difficult mixtures of pollutants.

Whole-transcriptome profiling using DNA microarrays has the advantage that the relative amount of transcripts from the whole genome may be easily determined compared to such techniques like proteomics. To understand the metabolism of bacteria in the rhizosphere, researchers have begun to utilize whole-genome profiling.

Although a tremendous amount of work remains to be performed, significant advances have been made through protein engineering and through metabolic engineering for the purposes of bioremediation. However, even though whole-transcriptome profiling and proteomics are utilized routinely in some disciplines, they remain to be utilized extensively in bioremediation. Furthermore, it is important to ensure engineered strains for field use are competitive; rhizoremediation can provide a niche for these engineered bacteria. Chromosomal integration of introduced genes can limit horizontal gene transfer to other species, but this should also be empirically verified to ensure that no adverse environmental effects occur.

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• Alpine microorganisms and low-temperature bioremediation
• Can fungi clean up radiation releases?
• Saturday Cinema: Bioremediation

Few bacteria are loners – more often they grow in crowds and squat on surfaces where they form a community. These so-called biofilms develop on any surface that bacteria can attach themselves to. The dilemma we face is that neither disinfectants and antibiotics, nor phagocytes (cells which typically destroy microorganisms) and our immune system can destroy these biofilms. This is a particular problem in hospitals if these bacteria form a community on a catheter or implant where they could potentially cause a serious infection. Scientists in Germany have just identified one of the fundamental mechanisms used by the bacteria in biofilms to protect themselves against the attacking phagocytes – the discovery that biofilm bacteria use chemical weapons to defend themselves.

Until now, scientists have been unable to understand the root of the biofilm problem – the inability of phagocytes to destroy these biofilms. The researchers investigated this problem by modelling the problem on marine bacteria. Marine bacteria face constant threats in their habitat from environmental phagocytes (amoebae) which behave in a similar way in the sea as do immune cells in our body (i.e. they seek out, and feed on, bacteria). As long as bacteria are swimming freely and separately in the water, they are easy pickings for these predators. However, if they become attached to a surface and socialize with other bacteria, the amoebae can no longer successfully attack them. The surprising thing was that the amoebae attacking the biofilms were de-activated or even killed. The bacteria are clearly not just building a fortress, they are also fighting back. The bacteria utilise chemical weapons to achieve this. A widespread and highly effective molecule used by marine bacteria is the pigment violacein. Once the defence system is ready, the biofilm shimmers a soft purple colour. If the attackers consume just a single cell of the biofilm – and the pigment they contain – this paralyses the attackers momentarily and the violacein triggers a suicide mechanism in the amoebae. Biofilms may no longer be seen just as a problem; they may also be a source of new bioactive agents. When organized in biofilms, bacteria produce highly effective substances which individual bacteria alone cannot produce. And the scientists hope to use these molecules to combat a specific group of pathogens, human parasites that cause devastating infections such as sleeping illness and malaria. Amoeba are ancient relatives of these pathogens and thus biofilm-derived weapons may provide an excellent basis for the design of new parasiticidal drugs.

Marine Biofilm Bacteria Evade Eukaryotic Predation by Targeted Chemical Defense. 2008 PLoS ONE 3(7): e2744
Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.

Related:

• Early bacterial adhesion dynamics
• Communication, cooperation, competition and cheating – bacteria are just like us
• Enzyme helps bacteria beat body by building biofilm
• Bacteria hedge their bets

When bacteria grow on solid surfaces, they can form three-dimensional communities called biofilms. Within these complex structures, bacteria can develop specific tolerance to different microbiocides, causing serious health and economic problems. Investigations of the key molecular events involved in biofilm formation have shown that surface-exposed adhesin proteins promote this process, but many questions remain regarding the mechanisms and biophysics of surface adhesion. A new paper introduces an original approach to investigating the very early steps in bacterial adhesion that uses dispersed colloidal surfaces as microbial adhesion substrates. Using flow cytometry, the authors performed a quantitative realtime analysis of adhesion kinetics of several strains of the bacterium Escherichia coli, which were genetically engineered to produce well-characterized cell-surface adhesins that are known to promote biofilm development. This provides evidence for previously unknown adhesin-dependent behaviors, such as clear-cut differences in the very initial phases of surface colonization, and demonstrates that initial adhesion correlates with almost instant surface property changes. Cell-to-cell association might serve as an amplification mechanism for surface colonization. This paper provides a new understanding of the intricate relationships between the physico-chemistry of abiotic surfaces and bacterial adhesion.

A short–time scale colloidal system reveals early bacterial adhesion dynamics. 2008 PLoS Biol 6(7): e167
The development of bacteria on abiotic surfaces has important public health and sanitary consequences. However, despite several decades of study of bacterial adhesion to inert surfaces, the biophysical mechanisms governing this process remain poorly understood, due, in particular, to the lack of methodologies covering the appropriate time scale. Using micrometric colloidal surface particles and flow cytometry analysis, we developed a rapid multiparametric approach to studying early events in adhesion of the bacterium Escherichia coli. This approach simultaneously describes the kinetics and amplitude of early steps in adhesion, changes in physicochemical surface properties within the first few seconds of adhesion, and the self-association state of attached and free-floating cells. Examination of the role of three well-characterized E. coli surface adhesion factors upon attachment to colloidal surfaces-curli fimbriae, F-conjugative pilus, and Ag43 adhesin-showed clear-cut differences in the very initial phases of surface colonization for cell-bearing surface structures, all known to promote biofilm development. Our multiparametric analysis revealed a correlation in the adhesion phase with cell-to-cell aggregation properties and demonstrated that this phenomenon amplified surface colonization once initial cell-surface attachment was achieved. Monitoring of real-time physicochemical particle surface properties showed that surface-active molecules of bacterial origin quickly modified surface properties, providing new insight into the intricate relations connecting abiotic surface physicochemical properties and bacterial adhesion. Hence, the biophysical analytical method described here provides a new and relevant approach to quantitatively and kinetically investigating bacterial adhesion and biofilm development.

Related:

• Enzyme helps bacteria beat body by building biofilm
• Bacterial Crowd Control
• Chinks in the Armour of Bacterial Biofilms
• Communication, cooperation, competition and cheating – bacteria are just like us

An antibody present in people with good oral health could become the first tool for dentists to assess a patient’s probable response to periodontal (gum) disease treatments. The antibody is to a protein called HtpG, made by the bacterium Porphyromonas gingivalis, an important pathogen in periodontal disease. The antibody also has potential as a vaccine candidate. Researchers discovered that the HtpG antibodies were present in much lower amounts in people with periodontal disease and in much higher concentrations in those with healthier teeth and gums. Typically, antibodies are elevated in people with disease, because they help fight the disease.
What has been seen in periodontal disease over the last 30-40 years is that patients with periodontal disease have higher levels of antibodies to the bacteria associated with periodontal disease, but these antibodies are not usually protective. The healthy patient makes high levels of the antibodies but to the right part of the organism. Not only were the HtpG antibodies present in higher amounts in people with healthier gums, those patients with the antibodies responded better to periodontal treatment.
The United States spends $8-$12 billion a year caring for people with serious periodontal disease. From a public health standpoint, it is important to identify those people who not only need therapy but will actually respond to a specific type of therapy. In the long run, this could lead to early interventional therapy to prevent periodontal disease from advancing, or even starting. The other part of the question is why people with periodontal disease do not make a good immune response to HtpG, and this could connect back to current thinking that oral health influences general health.

Serum Antibodies to Porphyromonas gingivalis Chaperone HtpG Predict Health in Periodontitis Susceptible Patients. 2008 PLoS ONE 3(4): e1984

The human vermiform (“worm-like”) appendix is a 5-10 cm long and 0.5-1 cm wide pouch that extends from the cecum of the large bowel. The architecture of the human appendix is unique among mammals, and few mammals other than humans have an appendix at all. The function of the human appendix has long been a matter of debate, with the structure often considered to be a vestige of evolutionary development despite evidence to the contrary based on comparative primate anatomy. The appendix is thought to have some immune function based on its association with substantial lymphatic tissue, although the specific nature of that putative function is unknown. Based (a) on a recently acquired understanding of immune-mediated biofilm formation by commensal bacteria in the mammalian gut, (b) on biofilm distribution in the large bowel, (c) the association of lymphoid tissue with the appendix, (d) the potential for biofilms to protect and support colonization by commensal bacteria, and (e) on the architecture of the human bowel, we propose that the human appendix is well suited as a “safe house” for commensal bacteria, providing support for bacterial growth and potentially facilitating re-inoculation of the colon in the event that the contents of the intestinal tract are purged following exposure to a pathogen.

Biofilms in the large bowel suggest an apparent function of the human vermiform appendix
J Theor Biol. 2007 249: 826-831

Related:

• Enzyme helps bacteria beat body by building biofilm
• Bacteria hedge their bets
• Bacterial Crowd Control
• Chinks in the Armour of Bacterial Biofilms

Bacteria are particularly harmful to human health when they band together to form a biofilm a sheet composed of many individual bacteria glued together because this can allow them to escape from both antibiotics and the immune system of their host. It is thought that most chronic infections are caused by bacterial biofilms, and a paper published in this week’s PLoS Biology explores the signalling system that causes bacteria to team up in this way.

Pseudomonas is a pathogen that forms biofilms in the lungs of people with cystic fibrosis. The new paper identifies a novel kind of control system for bacterial signalling. Bacteria form a biofilm when the concentration of a molecule called c-di-GMP gets above a certain threshold. Sondermann et al. have determined the structure of the enzyme that makes c-di-GMP. The enzyme is called WspR in Pseudomonas, and the way WspR is controlled in the cell is the focus of their paper. The authors determined the crystal structure of WspR and followed up with biochemical analysis of the enzyme. This work shows that WspR exists in an active form that produces c-di-GMP and is then bound by c-di-GMP and forced into an inactive form.

The study therefore reveals a finely balanced equilibrium between the synthesis and degradation of this key player in biofilm formation. New approaches to controlling the behavior of bacteria responsible for chronic infections can be envisaged. Because the signalling molecules involved in biofilm formation, such as c-di-GMP, are uniquely found in bacteria, the authors hope that there is potential for new therapeutic treatments based on this work – if you interrupted this bacterial signalling it would have no negative effect on the human host but could be devastating for the bacteria.

Phosphorylation-independent regulation of the diguanylate cyclase WspR
PLoS Biol 6(3): e67

Related:

• Chinks in the Armour of Bacterial Biofilms
• Who you gonna call? Slimebusters!
• Bacterial Crowd Control
• Quorum sensing in bacteria

On nutritional limitation, the bacterium Bacillus subtilis has the capability to enter the irreversible process of sporulation. This developmental process is bistable, and only a subpopulation of cells actually differentiates into endospores. Why a cell decides to sporulate or not to do so is poorly understood. Through the use of time-lapse microscopy, new research follows the growth, division, and differentiation of individual cells to identify elements of cell history and ancestry that could affect this decision process. These analyses show that during microcolony development, B. subtilis uses a bet-hedging strategy whereby some cells sporulate while others use alternative metabolites to continue growth, providing the latter subpopulation with a reproductive advantage.
B. subtilis is subject to aging. Nevertheless, the age of the cell plays no role in the decision of its fate. However, the physiological state of the cell’s ancestor (more than two generations removed) does affect the outcome of cellular differentiation. This epigenetic inheritance is based on positive feedback within the sporulation phosphorelay. The extended intergenerational “memory” caused by this autostimulatory network may be important for the development of multicellular structures such as fruiting bodies and biofilms.
The importance of epigenetic inheritance of cell fates on the population level may be based on the effect it has on neighboring cells. In bacterial colonies, which are sessile communities of cells, epigenetic inheritance affects those cells that are spatially grouped, in contrast to cells within planktonic cultures. The formation of biofilms requires systematic cell differentiation, and in B. subtilis, multicellular structure formation and sporulation are coordinated and intertwined by the action of Spo0A, suggesting that that epigenetic inheritance plays an important role in the formation of socially organized structures such as biofilms and fruiting bodies.

Bet-hedging and epigenetic inheritance in bacterial cell development
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0700463105