The Vibrio cholerae toxin co-regulated pilus (TCP) is a type 4b pilus that mediates bacterial microcolony formation, which is essential for intestinal colonization. Structural analyses have defined a surface domain of the TcpA pilin subunit that is displayed repeatedly around the pilus filament surface and forms the molecular basis for pilus-pilus interactions required for microcolony formation. The physical attributes of this domain that lead to pilus-pilus association between bacteria are not known. Mutational analysis has revealed alterations within this domain that allow pilus-pilus interactions among pili expressed by individual bacteria, but do not allow pilus-pilus mediated association between bacteria. We characterized these altered strains using conventional microscopy, as well as three-dimensional high-resolution field emission scanning electron microscopy (FESEM), to reveal the physical difference between nonproductive and productive pilus associations that lead to interactions among multiple bacteria and result in microcolony formation. These findings pave the way towards investigation of the biophysical parameters involved in this basic bacterial property that promotes colonization of intestinal and other biological surfaces. (C) 2011 Elsevier Inc. All rights reserved.