Biofilms in marine environments; implications for aquaculture, coral reefs, marine aquariums, and the human pathogen Vibrio cholerae Part 5
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.
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.