Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5-nucleotidase activity. The e (P4) protein is also shown to have NMN 5-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.Haemophilus influenzae, a gram-negative facultative anaerobic bacterium, is responsible for significant morbidity and mortality in young children (9, 35). In order to cultivate H. influenzae, complex medium is required, and if it is not blood based, it must contain two growth factors: nicotinamide adenine dinucleotide (NAD) and hemin (6). Early biochemical investigations established that nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can substitute for NAD, whereas nicotinamide, niacin, or other nicotine-based intermediates of the Preiss-Handler pathway cannot (10, 20, 31). The NAD dependency of H. influenzae was confirmed by the absence of the genes encoding the enzymes necessary for the de novo biosynthesis of NAD (8). Accumulation of nicotinamide nucleotides derived from NAD or NR has been demonstrated in H. influenzae and Haemophilus parainfluenzae (4, 11). For H. parainfluenzae the K m for transport is about 0.55 M for NAD and 0.14 M for NR, while the V max for NR is about four times that of NAD (4). This implies that NR is the substrate for an as-yet-unidentified inner membrane transporter, a proposal that is supported by the observation that NAD cannot be taken up into the cytosolic compartment as an intact molecule. Limited NAD salvage capacity resides within the H. influenzae cytosol, which can be demonstrated if cell extracts are incubated with NR or NMN, indicating the presence of an NMN adenylyl transferase or an NAD pyrophosphorylase activity (5, 16).

SummaryExogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy-dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5 H -nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD-nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5 H -nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild-type H. influenzae strains, when NADP was used as the sole source of nicotinamide-ribosyl.

One of the most common bacterially mediated diarrheal infections is caused by enterotoxigenic Escherichia coli (ETEC) strains. ETEC-derived plasmids are responsible for the distribution of the genes encoding the main toxins, namely, the heat-labile and heat-stable enterotoxins. The origins and transfer modes (intra-or interplasmid) of the toxin-encoding genes have not been characterized in detail. In this study, we investigated the DNA regions located near the heat-labile enterotoxin-encoding genes (eltAB) of several clinical isolates. It was found that the eltAB region is flanked by conserved 236-and 280-bp regions, followed by highly variable DNA sequences which consist mainly of partial insertion sequence (IS) elements. Furthermore, we demonstrated that rearrangements of the eltAB region of one particular isolate, which harbors an IS91R sequence next to eltAB, could be produced by a recA-independent but IS91 sequence-dependent mechanism. Possible mechanisms of dissemination of IS element-associated enterotoxin-encoding genes are discussed.Enterotoxigenic Escherichia coli (ETEC) infections are the major cause of bacterium-associated diarrheal diseases in developing countries (5, 15) and among travelers (4, 37). They are the subject of development of novel vaccines (19,45). The main virulence determinants of ETEC strains are heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST) (2, 40) and specific colonization factors (CFs) (13). ETEC strains colonize the small intestine and encode more than 20 different CFs (13). Indeed, combinations of CFs together with either ST or ST and LT in ETEC strains are the main risk factors for acquisition of acute ETEC-associated diarrheal diseases (14, 26).About one-third of the clinically relevant ETEC strains express both LT and ST, whereas the remaining two-thirds express either LT or ST (13). LT and ST can be encoded together or separately on large, variable plasmids called Ent plasmids (42), along with CFs, antibiotic resistance markers, and conjugation systems (11,41).Genes encoding LT (8) and cholera toxin (27) presumably have a common ancestor, since considerable amino acid and DNA sequence homologies are apparent (8). It has also been proposed that the LT genes are foreign genes which were acquired by horizontal gene transfer to form an enteropathogen (33, 50). The activities and structures of LT and cholera toxin are nearly identical. Both toxins consist of two subunits, the catalytically active subunit A and the receptor domain subunit B. The toxic activity of LT is caused by the catalytic activity of subunit A, which is able to catalyze the ADP-ribosylation of protein Gs␣ in eukaryotic cells. This in turn constitutively induces adenylate cyclase to produce elevated intracellular cyclic AMP concentrations (12).LT-encoding ETEC strains can be isolated from humans (LTh or LT-I) and animals (porcine LT [LTp] or LT-II) (2, 40). Although the overall similarity is high, some distinct differences at the DNA sequence level were observed (21, 47). The authors concluded...

The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3'-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3'-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm.

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