Biogenic polyamines (e.g., spermidine and spermine) are a group of essential polycationic compounds found in all living cells. The effects of spermine and spermidine on antibiotic susceptibility were examined with gram-negative Escherichia coli and Salmonella enterica serovar Typhimurium bacteria and clinical isolates of Pseudomonas aeruginosa and with gram-positive Staphylococcus aureus bacteria, including methicillin-resistant S. aureus (MRSA). Exogenous spermine exerted a dose-dependent inhibition effect on the growth of E. coli, S. enterica serovar Typhimurium, and S. aureus but not P. aeruginosa, as depicted by MIC and growth curve measurements. While the MICs of polymyxin and ciprofloxacin were in general increased by exogenous spermine and spermidine in P. aeruginosa, this adverse effect was not observed in enteric bacteria and S. aureus. It was found that spermine and spermidine can decrease the MICs of ␤-lactam antibiotics in all strains as well as other types of antibiotics in a strain-dependent manner. Significantly, the MICs of oxacillin for MRSA Mu50 and N315 were decreased more than 200-fold in the presence of spermine, and this effect of spermine was retained when assessed in the presence of divalent ions (magnesium or calcium; 3 mM) or sodium chloride (150 mM). The effect of spermine on the sensitization of P. aeruginosa and MRSA to antibiotics was further demonstrated by population analysis and time-killing assays. The results of checkerboard assays with E. coli and S. aureus indicated a strong synergistic effect of spermine in combination with ␤-lactams and chloramphenicol. The decreased MICs of ␤-lactams implied that the possible blockage of outer membrane porins by exogenous spermine or spermidine did not play a crucial role in most cases. In contrast, only the MIC of imipenem against P. aeruginosa was increased by exogenous spermine and spermidine, and this resistance effect was abolished in a mutant strain devoid of the outer membrane porin OprD. In E. coli, the MICs of carbenicillin, chloramphenicol, and tetracycline were decreased in two acrA mutants devoid of a major efflux pump, AcrAB. However, retention of the spermine effect on antibiotic susceptibility in two acrA mutants of E. coli suggested that the AcrAB efflux pump was not the target for a synergistic effect by spermine and antibiotics and ruled out the hypothesis of spermine serving as an efflux pump inhibitor in this organism. In summary, this interesting finding of the effect of spermine on antibiotic susceptibility provides the basis for a new potential approach against drug-resistant pathogens by use of existing ␤-lactam antibiotics.

Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for Helicobacter pylori infection.Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a rdxA gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant H. pylori, an intact rdxA gene has also been reported in Mtz-resistant H. pylori, suggesting that additional Mtz resistance mechanisms exist in H. pylori. We explored the nature of Mtz resistance among 544 clinical H. pylori isolates to clarify the role of rdxA inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in H. pylori. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to >256 g/ml. rdxA inactivation resulted in Mtz MICs of up to 32 g/ml for 6 Mtz-sensitive H. pylori strains and 128 g/ml for one Mtz-sensitive strain. Single or dual (with rdxA) inactivation of genes that encode ferredoxin-like protein (designated fdxB) and NAD(P)H flavin oxidoreductase (frxA) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance. fdxB inactivation resulted in a lower level of resistance than that from rdxA inactivation, whereas frxA inactivation resulted in MICs similar to those seen with rdxA inactivation. Further evidence for involvement of the frxA gene in Mtz resistance included the finding of a naturally inactivated frxA but an intact rdxA in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functional frxA genes, respectively, and transformation of an Mtz-resistant Escherichia coli strain to an Mtz sensitive strain by a naturally functional frxA gene but not an inactivated frxA gene. These results are consistent with the hypothesis that null mutations in fdxB, frxA, or rdxA may be involved in Mtz resistance.

Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of Pseudomonas aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as the precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into 4-aminobutyrate (GABA) and succinate before entering the tricarboxylic acid cycle in support of cell growth, as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were found to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown to be indispensable for polyamine utilization. The newly identified dadRAX locus encoding the regulator alanine transaminase and racemase coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintaining alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, an alternative ␥-glutamylation pathway for the conversion of putrescine into GABA is present in some organisms. Subsequently, GabD, GabT, and PA5313 were identified for GABA utilization. The growth defect of the PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA were also demonstrated in vitro. Polyamine utilization in general was proven to be independent of the PhoPQ two-component system, even though a modest induction of this operon was induced by polyamines. Multiple potent catabolic pathways, as depicted in this study, could serve pivotal roles in the control of intracellular polyamine levels.Agmatine, a cationic compound derived from arginine decarboxylation, serves as the precursor of three major polyamines, putrescine, spermidine, and spermine. These polyamines are the major organic polycations found in all living cells. Polyamines have pleiotropic effects on several cellular processes. In more complex organisms, these compounds are required for cell proliferation and differentiation (2). In Escherichia coli, these polycations play significant roles in the structural and functional organization of the chromosome (35). They are implicated in RNA synthesis through the stimulation of the activity of RNA polymerase and in protein synthesis through the stabilization of ribosomal structure and modulation of translational fidelity (9). In addition, polyamines are involved in the induction of recA in E. coli in response to UV or ␥ irradiation (14). Polyamines are thought to protect DNA from oxidative damage by serving as free radical scavengers (7, 13). Some microorganisms also use polyamines for the synthesis of se...

The galE gene product, UDP-galactose 4-epimerase, mediates the incorporation of galactose in extracellular polysaccharide materials such as the O-side chain of lipopolysaccharide (LPS). The O-side chain in H. pylori LPS has been shown to cross-react with Lewis x and/or y blood group antigens, suggesting its potential involvement in H. pylori-linked autoimmune disease. To study its role in H. pylori LPS biosynthesis, the galE gene was cloned, sequenced, and a galE-knockout H. pylori strain was constructed. The H. pylori galE gene encoded a protein of 344 amino acids with a molecular weight of 39K. The LPS profile from the galE-knockout H. pylori strain showed a lower molecular weight than that of the parental strain, indicating the involvement of the galE gene in LPS biosynthesis of H. pylori.

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD ؉ -dependent glutamate dehydrogenase (GDH), which converts L-glutamate, the product of the AST pathway, in ␣-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of ␤-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.The arginine succinyltransferase (AST) pathway (Fig. 1) is the major route for arginine catabolism under aerobic conditions in Pseudomonas aeruginosa, This pathway converts Larginine into L-glutamate with the concomitant release of three nitrogen moieties (11,13,14). Utilization of arginine as a carbon source entails deamination of glutamate to ␣-ketoglutarate, which is then channeled into the tricarboxylic acid (TCA) cycle. We have recently reported (18) the cloning and characterization of gdhB, which encodes a novel NAD ϩ -dependent glutamate dehydrogenase (NAD-GDH; GdhB). The expression of gdhB was shown to be inducible by exogenous arginine, and this induction was mediated by ArgR, the arginine regulatory protein. The activity of GdhB, a tetramer of equal 180-kDa subunits, was also found to be subject to allosteric activation by arginine. The induction of gdhB expression and the activation by arginine of the encoded enzyme clearly serve as mechanisms that coordinate aerobic utilization of arginine as a carbon source with glutamate utilization via the TCA cycle.The ArgR protein of P. aeruginosa does not exhibit any sequence homology to the arginine regulatory proteins from enteric bacteria (17,19) or Bacillus subtilis (5). Rather, ArgR of P. aeruginosa is a member of the AraC/XylS family of transcriptional regulators (27) and functions like other members of th...