Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of sixty -all infecting a common bacterial host -provides further insight into their diversity and evolution. Of the sixty phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, five of which can be further divided into subclusters; five genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the six genomes in cluster D share more than 97.5% average nucleotide similarity with each other. In contrast, similarity between the two genomes in Cluster I is barely detectable by diagonal plot analysis. The total of 6,858 predicted ORFs have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit smaller average size than genes of their host (205 residues compared to 315), phage genes in higher flux average only ∼100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.

Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electronelectron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. PP2A3 is a ubiquitous serine/threonine phosphatase involved in the regulation of numerous cell signaling pathways and cellular functions, including proliferation, cytoskeletal rearrangement, apoptosis, and cell migration (1-3). Several pathologies have been linked to dysregulation of PP2Ac, including Alzheimer disease, cancer, and diabetes (4 -8). The activity of PP2Ac is tightly controlled in vivo via association with regulatory subunits, interactions with other cellular proteins, and various post-translational modifications (9 -12). PP2A regulatory subunits play a critical role in determining phosphatase activity and substrate selectivity, as well as directing the subcellular localization of the PP2A holoenzyme (3). PP2A exists primarily as a heterotrimeric holoenzyme consisting of a structural A-subunit, a variable regulatory B-subunit, and PP2Ac. However, an atypical pool of PP2Ac exists in complex with the regulatory subunit Alpha4 that binds directly to PP2Ac in the absence of the A-and B-subunits (13-16). Recent studies have shown that Alpha4 plays a crucial role in the control of PP2A ubiquitination and stability (12,17,18).Alpha4, a multidomain protein with similarity to Tap42 from yeast, was initially discovered as a 52-kDa phosphoprotein in B-cell receptor complexes (16,19). Both Alpha4 and Tap42 consist of an N-terminal domain that contains the residues important for PP2Ac binding (20) and a C-terminal domain that is protease-sensitive and intrinsically disordered (21). The C-terminal domain of Alpha4 binds Mid1, a putative E3 ligase (12,22). Alpha4 regulates all three type 2A protein phosphatases (PP2Ac, PP4, and PP6), modulating both catalytic activity and expression levels (13,14,17,23). In addition to its association with PP2A family members, Alpha4 associates and co-localizes with Mid1, a putative E3 ubiquitin ligase thought to facilitate PP2Ac polyubiquitination (12,22). The C terminus of Alpha4 and the B-box1 domain of the Mid1 protein mediate the association between Mid1 and Alpha4 (12,22). Mutations in Mid1 have been linked to Opitz syndrome, a developmental disorder (24,25). At the cellular level, mutations in Mid1 lead to decreases in ubiquitination and degradation of PP2Ac, especially microtubule-associated PP2Ac (12, 26).Alpha4 serves as a scaffold for PP2Ac and Mid1 and protects PP2Ac fr...

In this report, a novel zymogram assay and coupled phosphoketolase assay were employed to demonstrate that Clostridium acetobutylicum gene CAC1343 encodes a bi-functional xylulose-5-P/fructose-6-P phosphoketolase (XFP). The specific activity of purified recombinant XFP was 6.9 U/mg on xylulose-5-P and 21 U/mg on fructose-6-P, while the specific activity of XFP in concentrated C. acetobutylicum whole-cell extract was 0.094 and 0.52 U/mg, respectively. Analysis of crude cell extracts indicated that XFP activity was present in cells grown on arabinose but not glucose and quantitative PCR was used to show that CAC1343 mRNA expression was induced 185-fold during growth on arabinose when compared to growth on glucose. HPLC analysis of metabolites revealed that during growth on xylose and glucose more butyrate than acetate was formed with final acetate:butyrate ratios of 0.72 and 0.83, respectively. Growth on arabinose caused a metabolic shift to more oxidized products with a final acetate:butyrate ratio of 1.95. The shift towards more oxidized products is consistent with the presence of an XFP, suggesting that arabinose is metabolized via a phosphoketolase pathway while xylose is probably metabolized via the pentose phosphate pathway.

VirA, an essential virulence factor in Shigella disease pathogenesis, is involved in the uptake, motility, and cell-to-cell spread of Shigella organisms within the human host. These functions have been attributed to a VirA protease activity and a mechanism of microtubule destruction via tubulin degradation. We report functional and crystallographic data indicating a novel VirA structure that lacks these activities but highlights the homology to the EspG virulence factor of pathogenic Escherichia coli.

Bacterial pathogens secrete effectors into their hosts that subvert host defenses and re-direct host processes. EspG is a type three-secretion effector with a disputed function that is found in enteropathogenic E. coli. Here we show: that EspG is structurally similar to VirA, a Shigella virulence factor; that EspG has a large, conserved pocket on its surface; that EspG binds directly to the amino-terminal inhibitory domain of human p21 activated kinase, (PAK); and that mutations to conserved residues in the surface pocket disrupt the interaction with PAK.Enteropathogenic E. coli, EPEC, are a leading bacterial cause of diarrhea, and the leading cause of diarrhea in infants (1). Infection occurs when, following ingestion by the host, EPEC adhere to intestinal epithelial cells and form attaching and effacing (A/E) lesions (2). The formation of A/E lesions requires the locus of enterocyte effacement (LEE), which encodes a type three-secretion system (TTSS) and multiple effector molecules (3). Shigella, a related genus of pathogens, use a similar TTSS to promote internalization, rather than A/E lesions (4). In both cases, effector proteins orchestrate a complex series of poorly understood molecular events to subvert host cell defenses, thereby preventing or delaying infection clearance.EPEC and Shigella express the homologous effector proteins, EspG and VirA, respectively. (EPEC also express EspG2, a distant, redundant homolog of EspG that is not LEE encoded (5).) These proteins are thought to have roles in cytoskeletal remodeling (6), although the mechanism of action and molecular targets are unknown. Recent reports indicate that these proteins do not act as papain family proteases (7,8) as was originally described (9-12). Despite ambiguity regarding the functional roles of EspG and VirA, it is clear that VirA has a role in cell-to-cell spread (13), and that EspG can reverse the spread phenotype of a virA null (14). The structure of VirA led to the identification of an inter-domain cleft and an inter-domain pocket (7,8). A goal for the present study was to determine which if either of these features was conserved in EspG and to gain insight into how these effectors modulate the host cytoskeleton.