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Xanthomonas albilineans is the causal pathogen of leaf scald disease of sugarcane. This devastating disease has been a major problem in most cane growing regions and continues to pose a worldwide economic threat to the sugarcane industry.Chlorosis inducing strains of the pathogen produce several toxic compounds. The major component, albicidin, inhibits DNA replication in sugarcane proplastids, resulting in blocked chloroplast development. Albicidin also inhibits prokaryotic DNA replication and is bactericidal to a range of Gram positive and Gram negative bacteria at concentrations as low as 1 ng ml-1. Albicidin has been partially characterized as a low molecular weight antibiotic that contains 38 carbon atoms with several aromatic rings. Albicidin deficient mutants of X. albilineans fail to cause chlorosis or any other symptom of leaf scald disease in inoculated sugarcane, indicating that albicidin phytotoxins play an import role in disease development. Therefore, resistance to albicidin may provide leaf scald disease resistance in sugarcane. X. albilineans is of interest as a potential source of these resistance genes. This project set out to investigate the genetics and mechanisms of albicidin resistance in X. albilineans.Two strategies were employed to isolate resistance genes from X. albilineans. No albicidin sensitive X. albilineans mutants were generated using transposon insertion mutagenesis; possibly because multiple redundant resistance genes contribute to albicidin resistance in X. albilineans, or because mutations in the resistance gene are lethal, even in Tox- mutants.The second strategy involved the construction of two X. albilineans genomic DNA cosmid libraries, followed by screening for clones conferring albicidin resistance in Escherichia coli. Albicidin resistant clones from the first library in E. coli DH5a proved unstable. One of these clones, E. coli JBDH93, conferred a 350-fold increase in albicidin resistance. However, the resistance gene was not transferrable on the cosmid. The mechanism of resistance did not involve export, binding, enzymatic detoxification or inactivation of the antibiotic, but did show a reduced uptake of the antibiotic. It is known that mutations in the tsx gene can prevent active uptake of the toxin in E. coli. Clone E. coli JBDH93 showed a reduced susceptibility to phage T6, which is characteristic of mutations in the tsx gene. PCR and Southern analysis showed that there had been some rearrangement in the tsx region. However, E. coli JBDH93 showed greater albicidin uptake than previously characterized albicidin resistant tsx mutants, and was resistant to approximately 10-fold higher albicidin concentration than previously characterized tsx mutants. After plating E. coli DH5a cells onto albicidin, no spontaneous mutants were obtained with albicidin resistance comparable to that of E. coli JBDH93. A gene from X. albilineans may be acting in combination with a tsx mutation to increase this clone's resistance to albicidin.The second genomic cosmid library was transformed into PMC 107, a multiple recombination deficient E. coli strain designed to minimise cosmid rearrangements. Several albicidin resistance clones were isolated. One of these clones showed at least a five-fold increased resistance to albicidin. The mechanism of resistance did not involve exclusion, binding, enzymatic detoxification or inactivation of the antibiotic. The 23 kb insert was subcloned and a putative 1487 bp albicidin resistance gene, designated albF was further isolated using PCR methods. In the vector pBlueScript II KS+ with induction of the lac promoter, only clones containing the ORF in the reverse orientation relative to the promoter could be generated, indicating that strong expression of albF is lethal to E. coli cells. Clones with the ORF in the forward orientation could only be maintained with repression of the lac promoter. Under conditions of lac repression, albF increased the minimum inhibitory concentration of albicidin by at least 3000-fold in the E. coli strains PMC107 and DH5a. No resistance to other antibiotics was observed.n n

OBJECTIVES To examine several poorly understood or contentious aspects of the antibacterial activity of silver (Ag(+)), including its cidality, mode of action, the prevalence of resistance amongst clinical staphylococcal isolates and the propensity for Staphylococcus aureus to develop Ag(+) resistance. METHODS The effects of Ag(+) on the viability, macromolecular synthesis and membrane integrity of S. aureus SH1000 were assessed using established methodology. Silver nitrate MICs were determined for a collection of staphylococcal isolates (n = 1006) collected from hospitals across Europe and Canada between 1997 and 2010. S. aureus biofilms were grown using the Calgary Biofilm Device. To examine the in vitro development of staphylococcal resistance to Ag(+), bacteria were subjected to continuous subculture in the presence of sub-MIC concentrations of Ag(+). RESULTS Silver was bactericidal against S. aureus in buffered solution, but bacteriostatic in growth medium, and was unable to eradicate staphylococcal biofilms in vitro. Challenge of S. aureus with Ag(+) caused rapid loss of membrane integrity and inhibition of the major macromolecular synthetic pathways. All clinical staphylococcal isolates were susceptible to ≤ 16 mg/L silver nitrate and prolonged exposure (42 days) to Ag(+) in vitro failed to select resistant mutants. CONCLUSIONS The rapid and extensive loss of membrane integrity observed upon challenge with Ag(+) suggests that the antibacterial activity results directly from damage to the bacterial membrane. The universal susceptibility of staphylococci to Ag(+), and failure to select for resistance to Ag(+), suggest that silver compounds remain a viable option for the prevention and treatment of topical staphylococcal infections.

International audience; Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis.

Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-l-Ala-d-Glu:meso-diaminopimelic acid (UDP-MurNAc-l-Ala-d-Glu:meso-A2pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that themurEgene fromChlamydia trachomatisis capable of complementing a conditionalEscherichia colimutant impaired in UDP-MurNAc-l-Ala-d-Glu:meso-A2pm ligase activity. Recombinant MurE fromC. trachomatis(MurECt) was overproduced in and purified fromE. coliin order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-l-Ala-d-Glu as the nucleotide substrate, MurECtdemonstrated ATP-dependentmeso-A2pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, thellandddisomers of A2pm,d-lysine) were also recognized by MurECt.However, the activities for these amino acid substrates were weaker than that formeso-A2pm. The specificity of MurECtfor three possibleC. trachomatispeptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relativekcat/Kmratios for UDP-MurNAc-l-Ala-d-Glu, UDP-MurNAc-l-Ser-d-Glu, and UDP-MurNAc-Gly-d-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid ismeso-A2pm. However, due to the lack of specificity of MurECtfor nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.

Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes (murA and murZ) encoding UDP-N-acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro, consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA, but remained almost unchanged following inactivation of murZ. Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA. Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.

Objectives The mechanisms by which antibiotics induce a post-antibiotic effect in susceptible bacteria are poorly understood. To explore the mechanisms more fully we examined the recovery of macromolecular synthesis in Escherichia coli during gentamicin- and rifampicin-induced post-antibiotic effects. Methods E. coli ATCC 25922 was exposed to rifampicin and to gentamicin at 5x MIC for 60 min to induce post-antibiotic effects. The antibiotics were then removed from the culture medium by washing the cells. The rates of DNA, RNA and protein synthesis during the post-antibiotic effect and recovery periods were subsequently determined by measuring the incorporation of radiolabelled uridine, thymidine and leucine into trichloroacetic acid precipitable material. Results Recovery of E. coli ATCC 25922 from the rifampicin-induced post-antibiotic effect coincided with the recovery of RNA and protein synthesis. Recovery from the gentamicin-induced post-antibiotic effect coincided with the recovery of protein synthesis. Conclusions These data support the hypothesis that antibiotic molecules retained in the cell mediate the post-antibiotic effect by suppressing the biochemical activity of their molecular targets.

Computer-based molecular design has been used to produce a series of new macrocyclic systems targeted against the bacterial cell wall biosynthetic enzyme MurD. Following their preparation, which involved a novel metathesis-based cyclisation as the key step, these systems were found to show good inhibition when assayed against the MurD enzyme.

BACKGROUND: ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl. METHODOLOGY/PRINCIPAL FINDINGS: Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli. CONCLUSIONS/SIGNIFICANCE: We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety.

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

The terminal dipeptide, D-Ala-D-Ala, of the peptidoglycan precursor UDPMurNAc-pentapetide is a crucial building block involved in peptidoglycan cross-linking. It is synthesized in the bacterial cytoplasm by the enzyme d-alanine:d-alanine ligase (Ddl). Structure-based virtual screening of the NCI diversity set of almost 2000 compounds was performed with a DdlB isoform from Escherichia coli using the computational tool AutoDock 4.0. The 130 best-ranked compounds from this screen were tested in an in vitro assay for their inhibition of E. coli DdlB. Three compounds were identified that inhibit the enzyme with K(i) values in micromolar range. Two of these also have promising antibacterial activities against Gram-positive and Gram-negative bacteria.

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add L-Ala, D-Glu, meso-A(2)pm or L-Lys, and D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute 'Diversity Set' on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC(50)=10 microM) and one novel MurF inhibitor (IC(50)=63 microM).

Objectives: Isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients are frequently hypermutable due to selection of mutants with defects in DNA repair genes such as mutS. Since P. aeruginosa grows as a biofilm within the infected CF lung, it is possible that this mode of growth enhances the mutability of the organism thereby increasing the opportunity to derive permanent hypermutators through mutation in DNA repair genes. We have now conducted experiments to examine this possibility. Methods: Using established procedures, we examined the mutability of P. aeruginosa PA01 in planktonic cultures and in biofilm cultures generated by growth in a Sorbarod system. Transcriptional profiling by DNA microarray was used to compare gene expression in planktonic and biofilm cells. Results: Mutation frequency determinations for resistance to rifampicin and ciprofloxacin demonstrated that biofilm cultures of P. aeruginosa displayed up to a 105-fold increase in mutability compared with planktonic cultures. Several genes (ahpC, katA, sodB and PA3529, a probable peroxidase) that encode enzymes conferring protection against oxidative DNA damage were down-regulated in biofilm cells. In particular, katA, which encodes the major pseudomonal antioxidant catalase, was down-regulated 7.7-fold. Conclusions: Down-regulation of antioxidant enzymes in P. aeruginosa biofilms may enhance the rate of mutagenic events due to the accumulation of DNA damage. Since P. aeruginosa forms biofilms in the CF lung, this mode of growth may enhance the direct selection of antibiotic-resistant organisms in CF patients and also increase the opportunity to derive permanent hypermutators thereby providing a further source of antibiotic-resistant mutants in the CF lung.

The mechanism of the postantibiotic effect (PAE) was examined in Escherichia coli . Drugs exhibited longer-lasting PAEs in an acrAB mutant, suggesting that intracellular drug concentrations influence the duration of the PAE. With specific assays for tetracycline and erythromycin, a direct link between intracellular persistence of antibiotics and maintenance of the PAE was established.

Sir, Chlamydophila pneumoniae (previously known as Chlamydia pneumoniae) was first characterized in 1986 and is now recognized as an important respiratory pathogen.1 The organism can cause pneumonia, bronchitis, sinusitis and pharyngitis as well as communityacquired pneumonia. 1 C. pneumoniae is susceptible in vitro to a number of antibiotics including macrolides, tetracyclines, quinolones and penicillins. 2,3 Although chlamydiae lack detectable quantities of peptidoglycan, the susceptibility of C. pneumoniae to penicillins is consistent with the presence of penicillin-binding proteins (PBPs) in this organism.4 In a recent study with Chlamydia trachomatis, we observed that mecillinam, a semi-synthetic penicillin, possessed potent in vitro anti-chlamydial activity that was superior to other penicillins, cephalosporins and carbapenems. 5 Since the susceptibility of C. pneumoniae to β-lactam antibiotics has not been extensively studied we decided to examine the activity of a number of these antibiotics, including mecillinam, against a recognized strain of C. pneumoniae. One strain was tested as there are only a few reference isolates and unlike C. trachomatis, there are no biovars or serovars of C. pneumoniae. C. pneumoniae strain ATCC VR1310 was cultured in HEp2 cells and antibiotic MICs and MBCs determined essentially as described previously.5 We observed that benzylpenicillin, ampicillin and mecillinam were the most active β-lactam antibiotics with MICs in the range 0.2–2.0 mg/L and MBCs in the range 0.2–8.0 mg/L (Table 1). Among these penicillins, mecillinam was the most potent, exhibiting an MIC/MBC of 0.2 mg/L. In contrast, ceftriaxone and meropenem only exhibited moderate activity and cefotaxime and imipenem were essentially inactive. These results with C. pneumoniae are qualitatively similar to those we previously reported for C. trachomatis. 5 Consequently, we have now demonstrated that

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP- N -acetylmuramate (UDP-MurNAc): l -alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli , since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli . It displayed in vitro ATP-dependent UDP-MurNAc: l -alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids ( l -alanine, l -serine, and glycine); comparable V max / K m values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.