Microorganisms attach to surfaces and develop biofilms. Biofilm-associated cells can be differentiated from their suspended counterparts by generation of an extracellular polymeric substance (EPS) matrix, reduced growth rates, and the up- and down- regulation of specific genes. Attachment is a complex process regulated by diverse characteristics of the growth medium, substratum, and cell surface. An established biofilm structure comprises microbial cells and EPS, has a defined architecture, and provides an optimal environment for the exchange of genetic material between cells. Cells may also communicate via quorum sensing, which may in turn affect biofilm processes such as detachment. Biofilms have great importance for public health because of their role in certain infectious diseases and importance in a variety of device-related infections. A greater understanding of biofilm processes should lead to novel, effective control strategies for biofilm control and a resulting improvement in patient management.

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Biofilms: microbial life on surfaces.

https://www.cell.com/trends/microbiology/pdf/S0966-842X(04)00264-1.pdf

Survival strategies of infectious biofilms.

Streptococcus mutans is the leading cause of dental caries (tooth decay) worldwide and is considered to be the most cariogenic of all of the oral streptococci. The genome of S. mutans UA159, a serotype c strain, has been completely sequenced and is composed of 2,030,936 base pairs. It contains 1,963 ORFs, 63% of which have been assigned putative functions. The genome analysis provides further insight into how S. mutans has adapted to surviving the oral environment through resource acquisition, defense against host factors, and use of gene products that maintain its niche against microbial competitors. S. mutans metabolizes a wide variety of carbohydrates via nonoxidative pathways, and all of these pathways have been identified, along with the associated transport systems whose genes account for almost 15% of the genome. Virulence genes associated with extracellular adherent glucan production, adhesins, acid tolerance, proteases, and putative hemolysins have been identified. Strain UA159 is naturally competent and contains all of the genes essential for competence and quorum sensing. Mobile genetic elements in the form of IS elements and transposons are prominent in the genome and include a previously uncharacterized conjugative transposon and a composite transposon containing genes for the synthesis of antibiotics of the gramicidin/bacitracin family; however, no bacteriophage genomes are present.

https://www.pnas.org/content/pnas/99/22/14434.full.pdf

Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen

Growth of oral bacteria in situ requires adhesion to a surface because the constant flow of host secretions thwarts the ability of planktonic cells to grow before they are swallowed. Therefore, oral bacteria evolved to form biofilms on hard tooth surfaces and on soft epithelial tissues, which often contain multiple bacterial species. Because these biofilms are easy to study, they have become the paradigm of multispecies biofilms. In this Review we describe the factors involved in the formation of these biofilms, including the initial adherence to the oral tissues and teeth, cooperation between bacterial species in the biofilm, signalling between the bacteria and its role in pathogenesis, and the transfer of DNA between bacteria. In all these aspects distance between cells of different species is integral for oral biofilm growth.

/pdf/oral-multispecies-biofilm-development-and-the-key-role-of-43nwhdhtx8.pdf

Oral multispecies biofilm development and the key role of cell–cell distance

Nearly 40 years ago, Dr. R.J. Gibbons made the first reports of the clinical relevance of what we now know as bacterial biofilms when he published his observations of the role of polysaccharide glycocalyx formation on teeth by Streptococcus mutans [Sci. Am. 238 (1978) 86]. As the clinical relevance of bacterial biofilm formation became increasingly apparent, interest in the phenomenon exploded. Studies are rapidly shedding light on the biomolecular pathways leading to this sessile mode of growth but many fundamental questions remain. The intent of this review is to consider the reasons why bacteria switch from a free-floating to a biofilm mode of growth. The currently available wealth of data pertaining to the molecular genetics of biofilm formation in commonly studied, clinically relevant, single-species biofilms will be discussed in an effort to decipher the motivation behind the transition from planktonic to sessile growth in the human body. Four potential incentives behind the formation of biofilms by bacteria during infection are considered: (1) protection from harmful conditions in the host (defense), (2) sequestration to a nutrient-rich area (colonization), (3) utilization of cooperative benefits (community), (4) biofilms normally grow as biofilms and planktonic cultures are an in vitro artifact (biofilms as the default mode of growth).

What drives bacteria to produce a biofilm

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm “lifestyle” for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.

Natural genetic transformation of Streptococcus mutans growing in biofilms.

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.

A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm Formation

PCR ligation mutagenesis in transformable streptococci: application and efficiency

Alterations in cellular growth are important in the progression of malignant disease. Cell growth regulation by tumour promoters can be complex. The present study demonstrates a novel link between ...

Tumour promoter mediated altered expression and regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in H-ras-transformed fibrosarcoma cell lines.

Inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation and occasionally, tumour cell proliferation is actually stimulated by transforming growth factor beta(1) (TGF-beta(1)). The present study demonstrates a novel link between alterations in TGF-beta(1) regulation during cellular transformation and malignant conversion and the expression of ornithine decarboxylase (ODC) which is a key rate limiting activity in the biosynthesis of polyamines and which is an enzyme that plays an important role in cell growth and differentiation. H-ras transformed mouse 10T(1/2) cell lines of varying degrees of malignant potential were examined for possible TGF-beta(1)-mediated alterations in ODC expression. Selective induction of ODC gene expression occurred. This induction was dependent upon the cellular phenotype expressed and the mechanisms responsible for the regulation of the TGF-beta(1)-mediated alterations in ODC expression varied as a function of malignant potential. The TGF-beta(1)-mediated alterations in ODC gene expression involves de novo protein synthesis, transcriptional, and post-transcriptional events. Evidence is also presented to suggest a possible role for protein kinase C-mediated events, protein phosphatases, and G-protein-coupled events in the TGF-beta(1)-mediated regulation of ODC expression in H-ras transformed cells. Evidence is also presented to suggest a possible role for cellular polyamines in the TGF-beta(1)-mediated alterations in ODC expression in H-ras transformed cells. Additionally, alterations in cellular polyamines were shown to influence TGF-beta(1) gene expression in H-ras transformed cells and that these alterations occurred, in part, through post-transcriptional events. The TGF-beta(1)-mediated regulation of ODC expression in H-ras transformed cells of varying degrees of malignant potential appears to be complex, multifaceted, and interactive. This study illustrates the importance of TGF-beta(1)-mediated regulation of ODC expression as a result of H-ras mediated cellular transformation and malignant progression, and further suggests that this TGF-beta(1)-mediated regulation constitutes an integral part of an altered growth regulatory program.

Janet H. Lee

Papers

Natural genetic transformation of Streptococcus mutans growing in biofilms.

A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm Formation

PCR ligation mutagenesis in transformable streptococci: application and efficiency

Tumour promoter mediated altered expression and regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in H-ras-transformed fibrosarcoma cell lines.

Transformation by H‐ras can result in aberrant regulation of ornithine decarboxylase gene expression by transforming growth factor‐β1