Morphogenesis of the T4 tail and tail fibers

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study

A cell surface display fluorescent biosensor for measuring MMP14 activity in real-time.

Despite numerous recent advances in imaging technologies, one continuing challenge for cell biologists and microscopists is the visualization and measurement of endogenous proteins as they function within living cells. Achieving this goal will provide a tool that investigators can use to associate cellular outcomes with the behavior and activity of many well-studied target proteins. Here, we describe the development of a plasmid-based fluorescent biosensor engineered to measure the location and activity of matrix metalloprotease-14 (MMP14). The biosensor design uses fluorogen-activating protein technology coupled with a MMP14-selective protease sequence to generate a binary, "switch-on" fluorescence reporter capable of measuring MMP14 location, activity, and temporal dynamics. The MMP14-fluorogen activating protein biosensor approach is applicable to both short and long-term imaging modalities and contains an adaptable module that can be used to study many membrane-bound proteases. This MMP14 biosensor promises to serve as a tool for the advancement of a broad range of investigations targeting MMP14 activity during cell migration in health and disease

Investigation of bacteriophage T4 by atomic force microscopy

Bacteriophage T4 was visualized using atomic force microscopy (AFM). The images were consistent with, and complementary to electron microscopy images. Head heights of dried particles containing DNA were about 75 nm in length and 60 nm in width, or about 100 nm and 85 nm respectively when scanned in fluid. The diameter of hydrated tail assemblies was 28 nm and their lengths about 130 nm. Seven to eight pronounced, right-handed helical turns with a pitch of 15 nm were evident on the tail assemblies. At the distal end of the tail was a knob shaped mass, presumably the baseplate. The opposite end, where the tail assembly joins the head, was tapered and connected to the portal complex, which was also visible. Phage that had ejected their DNA revealed the internal injection tube of the tail assembly. Heads disrupted by osmotic shock yielded boluses of closely packed DNA that unraveled slowly to expose threads composed of multiple twisted strands of nucleic acid. Assembly errors resulted in the appearance of several percent of the phage exhibiting two rather than one tail assemblies that were consistently oriented at about 72° to one another. No pattern of capsomeres was visible on native T4 heads. A mutant that is negative for the surface proteins hoc and soc, however, clearly revealed the icosahedral arrangement of ring shaped capsomeres on the surface. The hexameric rings have an outside diameter of about 14 nm, a pronounced central depression, and a center-to-center distance of 15 nm. Phage collapsed on cell surfaces appeared to be dissolving, possibly into the cell membrane

Three-dimensional structure of the bacteriophage P22 tail machine

The tail of the bacteriophage P22 is composed of multiple protein components and integrates various biological functions that are crucial to the assembly and infection of the phage. The three-dimensional structure of the P22 tail machine determined by electron cryo-microscopy and image reconstruction reveals how the five types of polypeptides present as 51 subunits are organized into this molecular machine through twelve-, six- and three-fold symmetry, and provides insights into molecular events during host cell attachment and phage DNA translocation

Unveiling an abundant core microbiota in the human adult colon by a phylogroup-independent searching approach

The potential presence of widespread and stable bacterial core phylogroups in the human colon has promoted considerable attention. Despite major efforts, no such phylogroups have yet been identified. Therefore, using a novel phylogroup- and tree-independent approach, we present a reanalysis of 1 114 722 V2 region and 71 550 near full-length 16S rRNA sequences from a total of 210 human beings, with widespread geographic origin, ethnic background and diet, in addition to a wide range of other mammals. We found two highly prevalent core phylogroups (cores 1 and 2), belonging to the clostridial family Lachnospiraceae. These core phylogroups showed a log-normal distribution among human individuals, while non-core phylogroups showed more skewed distributions towards individuals with low levels compared with the log-normal distribution. Molecular clock analyses suggest that core 2 co-evolved with the radiation of vertebrates, while core 1 co-evolved with the mammals. Taken together, the stability, prevalence and potential functionality support the fact that the identified core phylogroups are pivotal in maintaining gut homeostasis and health