Chung-Dar Lu

Polyamines Induce Resistance to Cationic Peptide, Aminoglycoside, and Quinolone Antibiotics in Pseudomonas aeruginosa PAO1

ABSTRACT Pseudomonas aeruginosa, a gram-negative bacterium of human pathogens, is noted for its environmental versatility, enormous metabolic capacity, and resistance to antibiotics. Overexpression of the outer membrane protein OprH and increased resistance to polycationic peptide antibiotics (e.g., polymyxin B) mediated by the PhoPQ two-component system on induction of a putative lipopolysaccharide (LPS) modification operon (PA3552-PA3559) have been reported as part of the adaptive responses to magnesium limitation in P. aeruginosa. Induction of the oprH-phoPQ operon and the LPS modification operon by exogenous spermidine was revealed from GeneChip analysis during studies of polyamine metabolism and was confirmed by the lacZ fusions of affected promoters. From the results of MIC measurements, it was found that addition of spermidine or other polyamines to the growth medium increased the MIC values of multiple antibiotics, including polycationic antibiotics, aminoglycosides, quinolones, and fluorescent dyes. MIC values of these compounds in the transposon insertion mutants of oprH, phoP, phoQ, and pmrB were also determined in the presence and absence of spermidine. The results showed that the spermidine effect on cationic peptide antibiotic and quinolone resistance was diminished in the phoP mutant only. The spermidine effect on antibiotics was not influenced by magnesium concentrations, as demonstrated by MICs and oprH::lacZ fusion studies in the presence of 20 μM or 2 mM magnesium. Furthermore, in spermidine uptake mutants, MICs of cationic peptide antibiotics and fluorescent dyes, but not of aminoglycosides and quinolones, were increased by spermidine. These results suggested the presence of a complicated molecular mechanism for polyamine-mediated resistance to multiple antibiotics in P. aeruginosa.

Functional Analysis and Regulation of the Divergent spuABCDEFGH-spuI Operons for Polyamine Uptake and Utilization in Pseudomonas aeruginosa PAO1

A multiple-gene locus for polyamine uptake and utilization was discovered in Pseudomonas aeruginosa PAO1. This locus contained nine genes designated spuABCDEFGHI (spu for spermidine and putrescine utilization). The physiological functions of the spu genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn5 transposon-mediated spu knockout mutants. Growth and uptake experiments support that the spuDEFGH genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that spuC encodes putrescine aminotransferase with pyruvate as the amino group receptor. Although spuA and spuB mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated. Assays of lacZ fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the spuABCDEFGH operon and the divergently transcribed spuI gene of unknown function. Since the observed induction effect of agmatine was abolished in an aguA mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the spuA-spuI divergent promoters. S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites. Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region. Finally, the absence of the putrescine-inducible spuA expression and putrescine aminotransferase (spuC) formation in the cbrB mutant indicated that the spu operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator.

Polyamine Effects on Antibiotic Susceptibility in Bacteria

ABSTRACT 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 β-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 β-lactams and chloramphenicol. The decreased MICs of β-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 β-lactam antibiotics.

Pathways and regulation of bacterial arginine metabolism and perspectives for obtaining arginine overproducing strains

Abstractl-Arginine is produced by bacterial fermentation and is consumed in food flavoring and pharmaceutical industries. A better understanding of arginine metabolism in bacteria could be beneficial for a rational design of recombinant l-arginine producers by genetic engineering. This mini-review illustrated the current status of genes and enzymes for arginine metabolism, including biosynthetic pathways, catabolic pathways, uptake and excretion systems, and regulation. The linkage of polyamine and glutamate metabolism to the arginine network was also discussed, followed by a perspective view on how to construct arginine overproducing strains of bacteria with increasing biosynthesis and excretion and decreasing catabolism and uptake.

Nonclassical Protein Secretion by Bacillus subtilis in the Stationary Phase Is Not Due to Cell Lysis

The carboxylesterase Est55 has been cloned and expressed in Bacillus subtilis strains. Est55, which lacks a classical, cleavable N-terminal signal sequence, was found to be secreted during the stationary phase of growth such that there is more Est55 in the medium than inside the cells. Several cytoplasmic proteins were also secreted in large amounts during late stationary phase, indicating that secretion in B. subtilis is not unique to Est55. These proteins, which all have defined cytoplasmic functions, include GroEL, DnaK, enolase, pyruvate dehydrogenase subunits PdhB and PdhD, and SodA. The release of Est55 and those proteins into the growth medium is not due to gross cell lysis, a conclusion that is supported by several lines of evidence: constant cell density and secretion in the presence of chloramphenicol, constant viability count, the absence of EF-Tu and SecA in the culture medium, and the lack of effect of autolysin-deficient mutants. The shedding of these proteins by membrane vesicles into the medium is minimal. More importantly, we have identified a hydrophobic α-helical domain within enolase that contributes to its secretion. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid gene-encoded mutant enolase is totally blocked, while the wild-type chromosomal enolase is secreted normally in the same cultures during the stationary phase, indicating differential specificity. We conclude that the secretion of Est55 and several cytoplasmic proteins without signal peptides in B. subtilis is a general phenomenon and is not a consequence of cell lysis or membrane shedding; instead, their secretion is through a process(es) in which protein domain structure plays a contributing factor.

Regulation of Carbon and Nitrogen Utilization by CbrAB and NtrBC Two-Component Systems in Pseudomonas aeruginosa

The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the DeltacbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.

Molecular Characterization and Regulation of the aguBA Operon, Responsible for Agmatine Utilization in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (M(r) 32,759; 292 amino acids) displayed sequence similarity (< or =60% identity) to enzymes of the beta-alanine synthase/nitrilase family. While the deduced AguA protein (M(r) 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (M(r) 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function.

Transcriptome Analysis of Agmatine and Putrescine Catabolism in Pseudomonas aeruginosa PAO1

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 gamma-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.

Polyamines Increase Antibiotic Susceptibility in Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen. Treatment is complicated by frequent acquired resistance to antipseudomonal therapies. Polyamines (cadaverine, putrescine, spermidine, and spermine) are ubiquitous polycationic compounds essential for all living organisms. In a dose-dependent manner, polyamines increased the susceptibility of P. aeruginosa to 14 β-lactam antibiotics, chloramphenicol, nalidixic acid, and trimethoprim as demonstrated by a reduction in MIC of up to 64-fold. This effect was partially antagonized (25 to 50%) by the presence of 10 mM of Mg2+ or Ca2+. In contrast, the effects of the outer membrane permeabilizers, polymyxin B nonapeptide and EDTA, were completely abolished by 3 mM Mg2+ or Ca2+. Changes on the outer membrane barrier by these compounds were assessed by activity measurements of periplasmic β-lactamase. The results showed that while EDTA and polymyxin B serve as outer membrane disorganizing agents as expected, exogenous spermidine and spermine did not exhibit any apparent effect on outer membrane permeability or rupture. In summary, these results strongly suggest that the increased antibiotic susceptibility by polyamines is exerted by a mechanism that differs from that of EDTA and polymyxin B. Polyamines might be potentially useful in antipseudomonal therapies by increasing the effectiveness of certain β-lactam antibiotics.

Transcriptome Analysis of the ArgR Regulon in Pseudomonas aeruginosa

Arginine metabolism in pseudomonads with multiple catabolic pathways for its utilization as carbon and nitrogen sources is of particular interest as the model system to study control of metabolic integration. We performed transcriptome analyses to identify genes controlled by the arginine regulatory protein ArgR and to better understand arginine metabolic pathways of P. aeruginosa. We compared gene expression in wild-type strain PAO1 with that in argR mutant strain PAO501 grown in glutamate minimal medium in the presence and absence of arginine. Ten putative transcriptional units of 28 genes were inducible by ArgR and arginine, including all known ArgR-regulated operons under aerobic conditions. The newly identified genes include the putative adcAB operon, which encodes a catabolic arginine decarboxylase and an antiporter protein, and PA0328, which encodes a hypothetical fusion protein of a peptidase and a type IV autotransporter. Also identified as members of the arginine network are the following solute transport systems: PA1971 (braZ) for branched-chain amino acids permease; PA2042 for a putative sodium:serine symporter; PA3934, which belongs to the family of small oligopeptide transporters; and PA5152-5155, which encodes components of an ABC transporter for a putative opine uptake system. The effect of arginine on the expression of these genes was confirmed by lacZ fusion studies and by DNA binding studies with purified ArgR. Only five transcriptional units of nine genes were qualified as repressible by ArgR and arginine, with three operons (argF, carAB, and argG) in arginine biosynthesis and two operons (gltBD and gdhA) in glutamate biosynthesis. These results indicate that ArgR is important in control of arginine and glutamate metabolism and that arginine and ArgR may have a redundant effect in inducing the uptake systems of certain compounds.