Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdx4, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdrA, pdxB, and pdxH but not in pdr. This observation suggested that pd4, like pdx4, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxj in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxj-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxj, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth ofpdrJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.

We report the first molecular genetic analysis of a pyridoxine 5'-phosphate oxidase, the PdxH gene product of Escherichia coli K-12. Chromosomal insertions in and around pdxH were generated with various transposons, and the resulting phenotypes were characterized. The DNA sequence of pdH was determined, and the promoters of pdxH and the downstream gene tyrS, which encodes tyrosyl-tRNA synthetase, were mapped by RNase T2 protection assays of chromosomal transcripts. These combined approaches led to the following conclusions: (i) pdxH is transcribed from a sigma 70-type promoter and shares its transcript with tyrS; (ii) tyrS is additionally transcribed from a relatively strong, nonconventional internal promoter that may contain an upstream activating sequence but whose expression is unaffected by a fis mutation; (iii) PdxH oxidase is basic, has a molecular mass of 25,545 Da, and shares strking homology (>40%o identity) with the developmentally regulated FprA protein of Myxococcus xanthus; (iv) mild pyridoxal 5'-phosphate limitation of pdxH mutants inhibits cell division and leads to formation of unsegregated nucleoids; (v) E. coli PdxH oxidase is required aerobically and anaerobically, but second-site suppressors that replace pdxH function entirely can be isolated; and (vi) pdrHx mutants excrete significant amounts of L-glutamate and a compound, probably at-ketoisovalerate, that triggers L-valine inhibition of E. coli K-12 strains. These findings extend earlier observations that pyridoxal 5'-phosphate biosynthetic and aminoacyl-tRNA synthetase genes are often members of complex, multifunctional operons. Our results also show that loss of pdxH function seriously disrupts cellular metabolism in unanticipated ways.Pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are important coenzymes that participate in many metabolic reactions, especially those involving amino acids (5, 22, 58). PLP and PMP are synthesized from pyridoxine (PN; vitamin B6), pyridoxal (PL), and pyridoxamine (PM) by phosphorylation and oxidation reactions catalyzed by PN kinase (PN/PL/PM kinase; pyridoxal kinase; EC 2.7.1.35) and PNP oxidase (PNP/PMP oxidase; pyridoxaminephosphate oxidase; EC 1.4.3.5), respectively ( Fig. 1) (15, 55). Of the three precursors, PN is thought to be the direct biosynthetic intermediate of PLP and is synthesized by bacteria, fungi, and higher plants (55). The biosynthesis of PN seems to occur by a branched pathway in Escherichia coli K-12 (33, 34) and most likely utilizes 4-hydroxythreonine (15, 33) and D-1-deoxyxylulose (23) as key intermediates. The last steps of PLP biosynthesis that take PN to PLP and PMP and interconvert the six B6 vitamers (Fig.

The massive increase of spam is posing a very serious threat to email which has become an important means of communication. Not only does it annoy users, but it also consumes much of the bandwidth of the Internet. Most spam filters in existence are based on the content of email one way or the other. While these anti-spam tools have proven very useful, they do not prevent the bandwidth from being wasted and spammers are learning to bypass them via clever manipulation of the spam content. A very different approach to spam detection is based on the behavior of email senders. In this paper, we propose a learning approach to spam sender detection based on features extracted from social networks constructed from email exchange logs. Legitimacy scores are assigned to senders based on their likelihood of being a legitimate sender. Moreover, we also explore various spam filtering and resisting possibilities.

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogentransport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants. Key words Light and metabolic regulation of genes involved in nitrogen metabolismIn non-legume plants, nitrogen assimilation begins with the uptake of inorganic nitrogen, nitrate or ammonium, from the soil.Nitrate is subsequently reduced to ammonium (NH 4 + ) by the sequential action of nitrate reductase and nitrite reductase (1,2). The resulting ammonium is then assimilated into an organic form as glutamate and glutamine. These amino acids are the nitrogen donors in the biosynthesis of essentially all