Abstract:We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12.54)-PSGH-(X31.54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structurefunction analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.Keywords: chloroperoxidase; homology; glucose-6-phosphatase; lipid phosphatase; motif; nonspecific acid phosphatase; phosphatase Phosphatases (phosphohydrolases) catalyze the hydrolysis of phosphoester and phosphoanhydride bonds of a diverse set of substrates including phosphorylated sugars, proteins, nucleic acids, and lipids (Boyer et al., 1961). Various criteria have been used to classify phosphatases into families. For example, phosphatases are classified on the basis of characteristics such as the molecular nature of the phosphate-containing substrate (phosphomonoester or phosphodiester), substrate type (proteinaceous or non-proteinaceous), substrate specificity (glucose-6-phosphatases, protein-tyrosine phosphatases), pH optimum (acid and alkaline phosphatases), size (high and low molecular weight phosphatases), and on the identity of the enzyme residue that is transiently phosphorylated during catalysis (histidine, serine, and cysteine phosphatases) (Boyer et al

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

In Saccharomyces cerevisiae, unsaturated fatty acids are formed from saturated acyl-CoA precursors by Ole1p, a ⌬-9 fatty acid desaturase. OLE1 mRNA levels are differentially regulated by the addition of saturated or unsaturated fatty acids to the growth medium. One component of this regulation system involves the control of OLE1 transcription. Saturated fatty acids induce a 1.6-fold increase in transcription activity, whereas a large family of unsaturated fatty acids repress OLE1 transcription as much as 60-fold. A deletion analysis of OLE1 promoter::lacZ fusion reporter genes identified a 111-base pair (bp) fatty acid-regulated (FAR) region approximately 580 bp upstream of the start codon that is essential for transcription activation and unsaturated fatty acid repression. Deletion of an 88-bp sequence within that region resulted in a complete loss in transcription activation and unsaturated fatty acid regulation. The 111-bp FAR element strongly activates transcription and confers unsaturated fatty acid regulation on a heterologous CYC1 promoter test plasmid. Essential elements required for unsaturated fatty acid repression of OLE1 were found in the 5 and 3 region of the 111-bp sequence. The FAR element-mediated activation and fatty acid repression of transcription was found to be closely tied to fatty acyl-CoA metabolism. Two fatty acid activation genes, FAA1 and FAA4, were found to be essential for unsaturated fatty acid repression of OLE1 through the FAR sequences. Disruption of either gene results in reduced levels of unsaturated fatty acid repression; disruption of both genes completely blocks the regulatory response. Acyl-CoA binding protein (ACBP) plays a role in determining the level of FAR element activated transcription. Disruption of the ACBP gene causes a >5-fold activation of OLE1 transcription and a similar increase in OLE1 mRNA levels. Unsaturated fatty acid repression of OLE1 transcription, however, is not affected by the disrupted ACBP gene. These studies show that promoter elements responsible for unsaturated fatty acid-mediated transcription repression are tightly linked to OLE1 activation sequences and that OLE1 transcription levels are closely tied to acyl-CoA metabolism.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.