Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism.

In enteric bacteria, the hexitol galactitol (Gat) (formerly dulcitol) is taken up through enzyme II (II(Gat)) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), and accumulated as galactitol 1-phosphate (Gat1P). The gat genes involved in galactitol metabolism have been isolated from the wild-type isolate Escherichia coli EC3132 and cloned on a 7.8-kbp PstI DNA fragment. They comprise six complete open reading frames and one truncated open reading frame in the order gatYZABCDR'. The genes gatABC code for the proteins GatA (150 residues) and GatB (94 residues), which correspond to the hydrophilic domains IIA(Gat) and IIB(Gat), and GatC, which represents a membrane-bound transporter domain IIC(Gat) (35 kDa, 427 residues). The three polypeptides together constitute a II(Gat) of average size (671 residues). Gene gatD codes for a Gat1P-specific NAD-dependent dehydrogenase (38 kDa, 346 residues), gatZ codes for a protein (42 kDa, 378 residues) of unknown function, and gatY (31 kDa, 286 residues) codes for a D-tagatose-1,6-bisphosphate aldolase with similarity to other known ketose-bisphosphate aldolases. The truncated gatR' gene, whose product shows similarity to the glucitol repressor GutR, closely resembles a gatR gene fragment from E. coli K-12. The gat genes map in both organisms at similar positions, in E. coli K-12, where they are transcribed counterclockwise at precisely 46.7 min or 2,173 to 2,180 kbp. The genes are expressed constitutively in both strains, probably due to a mutation(s) in gatR. Transcription initiation sites for the gatYp and the gatRp promoters were determined by primer extension analysis

Cloning of the Escherichia coli sor genes for L-sorbose transport and metabolism and physical mapping of the genes near metH and iclR.

The sor genes for L-sorbose (Sor) degradation of Escherichia coli EC3132, a wild-type strain, have been cloned on a 10.8-kbp fragment together with parts of the metH gene. The genes were mapped by restriction analysis, by deletion mapping, and by insertion mutagenesis with Tn1725. Seven sor genes with their corresponding gene products have been identified. They form an operon (gene order sorCpCDFBAME) inducible by L-sorbose, and their products have the following functions: SorC (36 kDa), regulatory protein with repressor-activator functions; SorD (29 kDa), D-glucitol-6-phosphate dehydrogenase; SorF and SorB (14 and 19 kDa, respectively), and SorA and SorM (27 and 29 kDa, respectively), two soluble and two membrane-bound proteins, respectively, of an L-sorbose phosphotransferase transport system; SorE (45 kDa), sorbose-1-phosphate reductase. The sor operon from E. coli EC3132 thus is identical to the operon from Klebsiella pneumoniae KAY2026. On the basis of restriction mapping followed by Southern hybridization experiments, the sor genes were mapped at 91.2 min on the chromosome, 3.3 kbp downstream of the metH-iclR gene cluster, and shown to be transcribed in a counterclockwise direction. The chromosomal map of the Sor+ strain EC3132 differs from that of the Sor- strain K-12 in approximately 8.6 kbp

NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs

A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, approximately 13% of the abundant sRNA RNAI is NAD-capped in the presence, and approximately 26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria