Adhesion proteins: structure-function relationships and role in bacterial colonization and biofilms.

细菌已经进化出复杂的适应性，以在特定的生态位中定居。它们可以表现为多细胞群体，相互合作以最大限度地提高成功率。作为生物膜，它们对根除具有显著的抗性。在这里，我们正在研究大的外膜蛋白（粘附素），其功能是粘附到细胞外基质和自粘附形成生物膜。 我们正在研究的几个例子之一是在海冰下发现的海洋细菌（Marinomonasprimoryensis）的1.5-MDa粘附素。在其C-末端，它具有RTX蛋白共有的I型分泌序列，并且在其旁边是冰结合结构域。另一端附着在细菌的外膜上。在它们之间是IgG样结构域的~120个相同重复，其将冰结合结构域从细菌表面突出。IgG重复序列的串联阵列以反平行方式彼此结合。因此，粘附素不仅将细菌与冰结合，而且将细菌聚集在一起以加强它们对冰的粘附。同样令人兴奋的是与冰结合结构域相邻的糖结合结构域的作用，它可能有助于将粘附素与生长中的生物膜中的相邻结构域结合。 这些RTX粘附素广泛分布于细菌中。我们将研究的一种是趋磁细菌，Magnetotacticum。 众所周知，这些细菌含有磁性晶体，可以帮助宿主找到低（但必需）氧气水平的最佳生态位。我们认为趋磁细菌通过它们的粘附素自我粘附并合作移动到最佳生态位。另一种目标细菌是Marinocarbonoclasticus，它能够在油和水之间的界面处形成生物膜以降解油。 在这里，我们将使用这些细菌作为模式生物，以制定在生物膜形成的早期阶段破坏自缔合的策略。我们将在定制设计的微流体室中通过显微镜监测细菌缔合。微流体设备将允许我们改变介质，形成引诱剂和排斥剂的梯度，并观察细菌与冰，油滴和纤维素等基质的关联。 该提案的一个组成部分将是对粘合中使用的每个域和域组合的结构分析。将生产重组蛋白用于结构和生物物理分析。功能分析将涉及诱变，结构域交换和删除实验，并转移粘附素基因到其他主机。 这些都是我们实验室擅长的技术和程序，并将成为我们三个研究生（T.万斯，C. Stevens和J. Arora）在我们的细菌粘附素计划中。当他们毕业时，他们将掌握这些生物化学和结构生物学方法，并有能力应用它们来解决复杂的生物学问题。

Bacteria have evolved sophisticated adaptations to colonize specific niches. They can behave as multicellular masses that cooperate with each other to maximize their success. As biofilms they are remarkably resistant to eradication. Here we are studying large outer membrane proteins (adhesins) that function in both adhesion to an extracellular matrix and self-adhesion to form biofilms. One of several example we are studying is a 1.5-MDa adhesin from a marine bacterium (Marinomonas primoryensis) found under sea ice. At its C-terminal end it has a Type I secretion sequence common to RTX proteins and next to it an ice-binding domain. The other end attaches to the bacterial outer membrane. In between are ~120 identical repeats of an IgG-like domain that project the ice-binding domain away from the bacterial surface. Tandem arrays of the IgG-repeats bind to each other in an antiparallel fashion. Thus the adhesin not only binds the bacteria to ice but also links bacteria together in clusters to strengthen their adhesion to ice. Equally exciting is the role of a sugar-binding domain adjacent to the ice-binding domain that may serve to help bind the adhesin to its neighbours in the growing biofilm. These RTX adhesins are widely distributed in bacteria. One we will study is from the magnetotactic bacterium, Magnetospirillum magnetotacticum. These bacteria are well known for containing magnetic crystals that help the host to find their optimal niche of low (but essential) oxygen levels. We suggest that the magnetotactic bacteria self-adhere through their adhesins and cooperate in moving to the optimal niche. Another target bacterium is Marinobacter hydrocarbonoclasticus, which is able to form biofilms at the interface between oil and water to degrade the oil. Here we will use these bacteria as model organisms to develop strategies for disrupting self-association in the early phases of biofilm formation. We will follow bacterial association in custom designed microfluidics chambers monitored by microscopy. The microfluidics apparatus will allow us to change media, form gradients of attractants and repellents, and observe bacterial associations with substrate like ice, oil droplets, and cellulose. An integral part of this proposal will be the structural analysis of each domain and domain combinations used in adhesion. Recombinant proteins will be produced for structural and biophysical analysis. Functional analysis will involve mutagenesis, domain swap and deletion experiments, and transfer of adhesin genes to other hosts. These are all techniques and procedures that our lab excels in and will be the basis for the training of our three graduate students (T.Vance, C. Stevens and J. Arora) in our bacterial adhesin program. By the time they graduate they will have command of these biochemistry and structural biology methodologies and the ability to apply them to solve complex biological questions.