GENETIC CONTROL OF BIOFILM FORMATION

Several genes and gene clusters have been identified in Vibrio cholerae that have aided in the understanding of how biofilms are formed and what environmental factors are determinants in their formation. The first gene involved is in the vps gene cluster. This gene cluster is involved in the creation of the VPS exopolysaccharide, a primary component in the biofilm matrix. The vps genes are clustered in two regions within the Vibrio cholerae genome, with vpsA-K in one region, and vpsL-q in another. Transposon inactivation mutants were screened, leading to the description of several of the genes in this cluster.

Although the function of vpsA and vpsL have not been fully described, mutations in these genes lead to a reversion from R-type colonies to L-type with low expression of VPS exopolysaccharide. . Several regulatory genes have also been described. VpsR is a positive regulator, and HapR and CytR are negative regulators. Recently, a second positive regulator has also been found, termed vpsT, it is a transcriptional activator that works in concert with vpsR to regulate each others expression, and both can also autoregulate their own expression. It is only with both of these genes activated that maximal biofilm is deposited. These regulatory genes show homology to typical two-component regulatory systems, although the sensor kinases have yet to be identified.

Utilizing a vpsL deletion mutant, it was also discovered that there are at least two pathways associated with biofilm production in Vibrio cholerae, and that they each respond to different environmental cues. These pathways were termed vps-dependent or vps-independent. The vps dependent pathway requires a nutrient rich media with the presence of pre-formed monosaccharides in the media. There is no calcium requirement for this pathway. The O-antigen is also required for vps-dependent formation. The vps independent pathway does not require the presence of monosaccharides in the media and can be expressed on minimal media, so long as milimolar amounts of calcium is present. It has been hypothesized that because Vibrio cholerae is associated with both marine and estuarine environments, different mechanisms are needed for survival in these environments.

THE VPS-DEPENDENT PATHWAY

The vps-dependent pathway is associated with nigh nutrient concentrations and is able to grow with low salinity and low calcium concentrations, as one would expect during rainfall and runoff entering a positive estuary. Nutrient concentrations are highly variable in this environment and the biofilm may even play a role in the accumulation and storage of nutrients for starvation conditions. On the other hand, the vps-independent pathway only has a requirement for calcium. This less selective pathway would be expressed in the marine environment, where vibrios are often found colonizing nutrient sources such as the chitinous exoskeletons of planktonic organisms.

An additional gene has been found that plays a role in producing the proper structures for biofilm development. It has been termed mbaA, for maintenance of biofilm architecture. In vps-dependent biofilm formation, the vps gene clusters are activated by the presence of a mannose-sensitive hemagglutinin type IV pilus (MSHA). Once initial attachment has occurred, the EPS is synthesized as the cells move via their flagella, and additional planktonic cells are recruited for settlement. The gene mbaA is not associated with these early stages of biofilm development. Instead, they appear to be regulators that control the amount of matrix EPS that is being produced in mature biofilms.

To find the mbaA gene, a mini-Tn10 mutant was isolated by researchers from a screen that produced abnormally robust biofilms. The insertion was determined to be at the 315th codon in a 2,376-bp open reading frame with no determined function. Sequence analysis indicated that this is likely a three-gene operon. Based on the identification of this mutant, an mbaA deletion mutant was created. These mutants showed extremely high levels of EPS being secreted, and none of the biofilms had the peaks, valleys, and channels associated with mature biofilms. Although the biofilms being formed were vigorous, when mbaA mutants were constructed with defects in either the MSHA or flagella genes, no biofilms were formed. This indicates that early requirements for biofilm formation are not circumvented by the mbaA mutation, and its presence occurs later in formation. Similarly, deletions in the vps gene clusters that are associated with defects in biofilm formation were not overcome by the mbaA deletion. It was also determined that the increased EPS production was not associated with either increased cell density or cell division, again supporting the hypothesis that mbaA is a regulator of EPS production.

SUMMARY

Based on these findings, it is apparent that biofilm formation is a critical factor in the environmental survival of Vibrio cholerae, and is likely a strong determinant of pathogenesis in many primarily marine pathogens, especially those associated with coral beaching, wound infections of fish, and secondary infections following parasite attack. The presence of two distinct and independently regulated pathways for the formation of these structures demonstrates their importance. With different structural types created in response to varying environmental conditions, these biofilms are able to survive starvation conditions, create ideal conditions for exchange of genetic information via horizontal gene transfer, and maximize survival when attached to the exoskeletons of planktonic crustaceans for long-term oceanic survival and dispersal. Similarly, survival within a biofilm while attached to the chitinous exoskeletons might facilitate transport and survival through the GI tract when ingested by larger organisms, allowing the possible infection of a new host.

In the next sections, we will look at how biofilms may aid in coral pathogenesis, aquaculture mortalities, and filtration systems.

BIOFILMS ASSOCIATED WITH Vibrio spp. BACTERIA

In the first sections we looked at a survey of the attributes of biofilms as survival mechanisms and as generalized virulence attributes. In this section, we will be examining the molecular mechanisms that control biofilm formation, and the role of these mechanisms in the pathogenesis of Vibrio species bacteria. Vibrios are found in nearly every marine aquarium, and are opportunistic pathogens of many ornamental fishes. It is also becoming increasingly evident that Vibrios are responsible for a variety of coral diseases, and are also potential pathogens of human reefkeepers. As such, their effects in aquarium settings are being studied in great detail. Some bits of this section do tend to get a bit thick, but I will attempt to sum all of this up at the end.

The genus Vibrio is a diverse group of bacteria found in myriad microcosms within the marine environment. Vibrios have been associated with disease in intensive aquaculture systems, coral bleaching, they are even found in the light organs of marine fishes and are a resident of the teeth of the great white shark. The best-known member of this group is Vibrio cholerae, the causative agent of human cholera. This is a classic disease of mankind and continues to be a significant cause of morbidity and mortality in developing nations.

Unlike other pathogens that utilize biofilms to evade the host immune response, biofilms do not appear to play a role in the disease cholera. This infection is defined by toxin production and does generally have a carrier or chronic state. Instead, toxin production causes fluid production causing copious diarrhea and associated release of the bacteria back into the environment. As a self-limiting disease, cholera would have little use for biofilm genes in the host. However, these bacteria are strongly associated with biofilm development in the marine environment.

Vibrio cholerae can assume two forms when plated on laboratory media. The most common form is the smooth or luminescent (L) form. Occasionally colonies assume a wrinkled or rugose (R) form. It appears that the rugose form is actually a biofilm form. The smooth form is typically isolated from patients infected with Vibrio cholerae. When grown on media that stresses the bacteria, L colonies become R colonies at a high rate. When these R colonies are placed back on rich media, they revert to an L form at a rate of 1.5X10-5. When these bacteria were compared using fingerprint analysis, they were found to be genetically identical. This indicates that the bacteria are not distinct genotypes, but rather undergo a phase variation triggered by stress through an unidentified mechanism.

BIOFILMS IN ENVIRONMENTAL SURVIVAL

Although understanding the roles of biofilms in human pathogenesis is of obvious importance, understanding the roles of environmental biofilms may be even more beneficial when considered in an economic, industrial, and medical perspective. Biofilms have been shown to be ubiquitous in aquatic environments. They have tremendous importance in sewage treatment, bioreactors, nutrient cycling, and the biogeographical transformation of carcasses including the progression of marine snow. They also allow survival in extreme environments such as acidic mine effluent, hypersaline environments, even the lake ice of Antarctica.

These environmental survival attributes make it difficult to remove biofilm bacteria in environments such as drinking water systems where disinfection is of critical importance. Biofilms make the bacteria resistant to common disinfectants such as chlorine, acids, bases, and heavy metals. When biofilms develop in industrial or potable water systems, removal is a difficult and costly endeavor.

Next will be a detailed examination of the molecular mechanisms and regulation of biofilms in marine microbes with an emphasis on those pf the genus Vibrio, a group of pathogens that can affect organisms ranging from corals to humans.

Following this (thick) section will look at the role of biofilms in aquaculture operations and marine aquariums with respect to filtration, fish health, and coral diseases.