Relaxed mutants ofSerratia marcescens SM-6. Biochemical traits and relevance of therel + allele for the formation of exoenzymes

Serratia marcescens SM-6 when starved for a required amino acid stops synthesizing protein and RNA and accumulates two nucleotides which cochromatograph with ppGpp and pppGpp. These features are characteristic of bacterial strains with stringent RNA control (rel ⁺). Two independent mutants were isolated which resemble relaxed (relA) mutants ofEscherichia coli; they continue to synthesize RNA and accumulate neither ppGpp nor pppGpp when deprived of the required amino acid. The extracellular enzyme activities (nuclease, protease, lipase) of the relaxed mutants are about the same as those of the parental stringent strain when studied under standard growth conditions. Exoenzyme-deficient (nuc; prt) and exoenzyme-hyperproducing (nuc ^su) mutants were isolated from both stringent and relaxed strains ofS. marcences SM-6 and no change of the cellular ability to form ppGpp and pppGpp could be observed. From these results it appears that the formation of exoenzymes ofS. marcescens SM-6 is independent of stringent/relaxed RNA control.Abbreviations cpd cyclic nucleotide phosphodiesterase deficient - nuc nuclease deficient - nuc ^su nuclease hyperproducing - prt protease deficient - rel relaxed control - spo ppGpp deficient (spot less) - ppGpp guanosine tetraphosphate - pppGpp guanosine pentaphosphate - TCA trichloroacetic acid - OD optical density - EU enzyme units     

Synthetic analogs of anoplin show improved antimicrobial activities

We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin‐resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin‐resistant Enterococcus faecium (ATCC 700221) (VRE), and Candida albicans (ATCC 200955). The anoplin analogs contain substitutions in amino acid positions 2, 3, 5, 6, 8, 9, and 10. We use these peptides to study the effect of altering the charge and hydrophobicity of anoplin on activity against red blood cells and microorganisms. We find that increasing the charge and/or hydrophobicity improves antimicrobial activity and increases hemolytic activity. For each strain tested, we identify at least six anoplin analogs with an improved therapeutic index compared with anoplin, the only exception being Enterococcus faecium, against which only few compounds are more specific than anoplin. Both 2Nal⁶ and Cha⁶ show improved therapeutic index against all strains tested. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Siderophore production and utilization byRhizobium trifolii

Summary Several strains ofRhizobium trifolii were tested for their ability to synthesize and utilize phenolate or hydroxamate types of siderophores. None of the nodulating strains ofR. trifolii was able to produce detectable amounts of siderophores. Only the non-nodulating strainR. trifolii AR6 formed a phenolate siderophore, which stimulated the growth of the siderophore-negative mutant AR65. Other strains ofR. trifolii could not utilize iron from exogenously supplied Desferal, pseudobactin or citrate. The siderophore fromR. trifolii AR6 and 2,3-dihydroxybenzoic acid slightly stimulated the growth of someR. trifolii strains.     

Influence of cultural conditions and mutations on the composition of the outer membrane proteins ofEscherichia coli

Summary VariousEscherichia coli strains differ in the composition of their major outer membrane proteins. However, allE. coli K12 strains tested possess the same major outer membrane proteinsa, b, c andd although quantitative differences were detected.The influence of growth conditions on the composition of the major outer membrane proteins ofE. coli was analyzed. It was found that neither the growth phase at which the cells are harvested, nor the fatty acid composition of the phospholipids has a considerable influence on the composition of these proteins. However, the composition of the growth medium, and, to a less extent, the growth temperature, have a pronounced influence.Certain mutants, changed in the composition of their lipopolysaccharide, are deficient in proteinb. Also mutants deficient in proteinc andd respectively, are described.Proteinsb andc ofE. coli K12 were found to be associated with peptidoglycan. Protein bands, corresponding with flagellin and pilin respectively, were identified.     

Formation of biologically active substances by rhizosphere bacteria and their effect on plant growth

Nine out of seventeen strains of bacteria with a pronounced effect on seed germination and on seedling growth, isolated from root surfaces and rhizosphere soil of maize, were selected for a study on the formation of biologically active substances. β-Indole acetic acid (45–72 μg/1.000 ml) was produced by four strains, gibberelline-like substances (1.0–60.0 μg/1.000ml) by all strains, biotin and pantothenic acid by the majority of strains and nicotinic acid by five strains. Amino acids were formed by all strains but in low amounts. Four strains produced growth inhibitors. The highest amounts of biologically active substances were found in cultures ofPseudomonas fluorescens andBacillus brevis. The various cultures ofPseudomonas fluorescens differed in their capability to produce biologically active substances. The majority of bacterial cultures or their supernatants significantly stimulated the germination of seeds and some of them significantly affected the growth of plants. Inoculation of maize seeds with strainsPseudomonas fluorescens andChromobacterium violaceum significantly increased the yield of dry matter of plants.     

Response of RP1+ and RP1− strains ofEscherichia coli to antibacterial agents and transfer of resistance toPseudomonas aeruginosa

The sensitivity of strains ofEscherichia coli, with and without the RP1 R-factor, to antibiotics and other antibacterial agents has been studied. RP1⁺ strains ofE. coli were resistant to kanamycin, carbenicillin, and tetracycline, resistance to the first two antibiotics being produced by destruction of the drugs. This resistance could be transferred to two strains ofPseudomonas aeruginosa. The parent strain ofE. coli UB 1005, its two mutant strains (DC2 and DC3), and two of the strains with the RP1 R-factor showed a similar order of sensitivity to phenylmercuric nitrate, chlorhexidine, thiomersal, and mercuric chloride.E. coli strains DC2 and DC2 (RP1⁺) were the most sensitive to benzalkonium chloride and cetrimide. RP1⁺ strains were more resistant than RP1⁻ strains to lysozyme-ethylenediaminetetraacetic acid, but treatment of the former strains with acriflavine rendered the cells more sensitive to the lytic system. There was no evidence thatP. aeruginosa (RP1⁺) strains possessed increased resistance to polymyxin or to disinfectants, although they became somewhat less sensitive to lysozyme-ethylenediaminetetraacetic acid.     

Coliforms and enterococci isolated from the intestinal tract of conventional mice

Coliforms and enterococci were isolated from the intestinal tract of infant (12-day-old) and adult (6-to 8-week-old) conventional mice. Eighty coliform isolates and eighty enterococcal strains were grouped according to their ability to ferment or hydrolyze various substrates. Sixty-one of the coliform isolates were identified asEscherichia coli. The remaining 19 strains were similar toE. coli, but did not produce-galactosidase. The enterococci belonged to two species:Streptococcus faecium andS. faecalis. Four biotypes ofS. faecium and two biotypes ofS. faecalis were detected. Xylosefermenting enterococci were isolated with a higher frequency from infant mice than from adults.     

An adhesive factor found in strains ofEscherichia coli belonging to the traditional infantile enteropathogenic serotypes

Escherichia coli strains isolated from outbreaks of diarrheal disease were tested for the presence of adhesive factors. Fifty-one of these strains belonged to traditional infantile entero-pathogenic serotypes (EPEC) and 17 belonged to other serotypes. None of these strains were enterotoxigenic and none possessed colonization factors CFA/I or CFA/II, which have been described among strains of enterotoxigenicE. coli (ETEC). EnterotoxigenicE. coli strains from patients with diarrhea and strains which were neither EPEC nor ETEC from subjects without diarrhea were also examined. By means of a tissue culture technique using HEp-2 cells, a new adhesive factor was found to occur with greater frequency in EPEC strains. The adhesive factor was found less frequently in the other groups ofE. coli studied. It was distinct from type 1 pili and was not inhibited by the presence ofD-mannose.     

Phage cocktail strategies for the suppression of a pathogen in a cross-feeding coculture

Cocktail combinations of bacteria-infecting viruses (bacteriophages) can suppress pathogenic bacterial growth. However, predicting how phage cocktails influence microbial communities with complex ecological interactions, specifically cross-feeding interactions in which bacteria exchange nutrients, remains challenging. Here, we used experiments and mathematical simulations to determine how to best suppress a model pathogen, E. coli, when obligately cross-feeding with S. enterica. We tested whether the duration of pathogen suppression caused by a two-lytic phage cocktail was maximized when both phages targeted E. coli, or when one phage targeted E. coli and the other its cross-feeding partner, S. enterica. Experimentally, we observed that cocktails targeting both cross-feeders suppressed E. coli growth longer than cocktails targeting only E. coli. Two non-mutually exclusive mechanisms could explain these results: (i) we found that treatment with two E. coli phage led to the evolution of a mucoid phenotype that provided cross-resistance against both phages, and (ii) S. enterica set the growth rate of the coculture, and therefore, targeting S. enterica had a stronger effect on pathogen suppression. Simulations suggested that cross-resistance and the relative growth rates of cross-feeders modulated the duration of E. coli suppression. More broadly, we describe a novel bacteriophage cocktail strategy for pathogens that cross-feed.     

Interaction of lead nitrate and cadmium chloride withEscherichia coli K-12 andSalmonella typhimurium global regulatory mutants

Summary To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb²⁺ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO₃)₂ were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO₃)₂) and CdCl₂. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.     

Isolation of a catabolic product of 2-butynedioic acid that acts as a precursor of nicotinamide adenine dinucleotide

The de novo nicotinamide adenine dinucleotide biosynthetic pathway ofEscherichia coli was investigated; using a cell-free extract of anadB mutant, we synthesized a precursor of quinolinic acid, a key intermediate in the pathway, from¹⁴C-aspartic acid. The synthesis of this compound was repressible by nicotinic acid. This compund was partially purified by ion-exchange column chromatography and then further purified and characterized by ascending partition chromatography on thin-layer plates. A metabolic scheme is presented which hypothesizes that the isolated precursor of quinolinic acid results from the amination of 2-butynedioic acid.     

Inhibition of growth ofLegionella species by heterotrophic plate count bacteria isolated from chlorinated drinking water

The ability of heterotrophic plate count bacterial strains isolated from chlorinated drinking water on low-nutrient media to inhibit the growth ofLegionella species was examined. Between 16% and 32% of these strains were able to inhibit the growth ofLegionella species when tested on buffered charcoal yeast extract agar. The exact proportion of inhibiting strains varied with the individualLegionella species. Two strains that inhibited the growth of severalLegionella species could also stimulate the growth of the same species when both the test strain and theLegionella species were grown on buffered charcoal yeast extract agar that lacked the essential amino acidl-cysteine.     

Nutritional requirements ofCytophaga johnsonae and some of its auxotrophic mutants

A defined medium for growing cells ofCytophaga johnsonae was developed for use in studies of the genetics of gliding motility of this organism. The defined medium, containing only mineral salts and any of six sugars, supports good growth of actively motile cells of several strains ofC. johnsonae. Characteristics of growth in the defined medium and ability of cells to use alternative sources of carbon and nitrogen are reported. Certain compounds, when added to solid media, inhibit colony spread ofC. johnsonae without affecting motility of individual cells. Amino acid requirements were determined for 41 of 43 auxotrophic mutants isolated following penicillin selection of unmutagenized cells growing in the minimal medium.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Mannityl opine analogs allow isolation of catabolic pathway regulatory mutants

Five virulent Agrobacterium spp. strains that can catabolize the mannityl opines mannopine (MOP), mannopinic acid ( MOA ), and agropinic acid (AGA) were tested for their ability to grow on analogs of these compounds. Analogs containing alternative amino acids replacing glutamic acid or glutamine were generally refused by these bacteria, but mutants were obtained that catabolized the entire family of analogs. In the case of strain C58C1 (pRi 8196), we demonstrated that typical mutants were constitutive for MOP uptake, whereas the wild-type parent was inducible by MOP. Analogs of MOA prepared from a variety of sugars instead of mannose were generally refused, except for a strain carrying pTi B6-806, which grew well on all such analogs. The analogs allowed selection of mutants of all strains. Although most wild-type strains were inducible for AGA uptake, typical mutants selected from strain C58C1 (pRi 8196) were found to be constitutive for uptake of AGA, as was the wild-type strain carrying pTi B6-806. Such constitutive mutants grew on all sugar analogs of MOP, MOA , and AGA tested. The pTi B6-806-containing strain was tested for growth on a more extended series of analogs, including tetrose , triose, diose , and disaccharide analogs, all of which were accepted. Only ketose analogs were refused. Selection of promiscuous regulatory mutants by the two types of opine analogs suggests that the repressor proteins of MOP and AGA permease/ catabolase systems are chiefly responsible for the specificity of the pathways.     

COMPARATIVE SENSITIVITY OF THREE SPECIES OF CHLAMYDOMONAS TO ANALOGS OF METABOLITES1,2

Twenty-five analogs of metabolites were tested for their ability to inhibit the growth of Chlamydomonas eugametos, C. moewusii, and C. reinhardii. Eleven of the tested analogs were inhibitory to 1 or more species, but only 3 compounds were inhibitory to all 3.     

Incidence of antimicrobial resistance among Enterobacteriaceae isolated in Riyadh

The antimicrobial resistance of 1,018 isolates of Enterobacteriaceae isolated from fecal specimens of the urban population of Riyadh, Saudi Arabia, was studied. Resistance to 1 or more of 10 antimicrobial agents was encountered in 50.2% of the isolates. Of the isolates tested,Escherichia coli (0.8%) andKlebsiella species (1.6%) were found resistant to seven antimicrobial agents simultaneously: ampicillin, chloramphenicol, kanamycin, colistin, streptomycin, tetracycline, and carbenicillin. Resistance to nalidixic acid was encountered in only 0.68% of theE. coli isolates. No isolate was found to be resistant to gentamicin. Eighty-six of the resistant strains were tested for their ability to transfer their resistance. Forty percent were able to do so withE. coli K-12.     

Utilization of the tricarboxylic acid cycle intermediates and symbiotic effectiveness inRhizobium meliloti

Summary The utilization of the tricarboxylic acid cycle intermediates and related compounds was studied in strains ofRhizobium meliloti having different symbiotic effectiveness. In general, the very effective (VE) strains used these compounds as sole carbon source better than the ineffective (I) strains. However, a significant different was observed between VE and I strains in their ability to use acetate or oxaloacetate for growth. In fact, at a concentration of 2 mM, 80% of the VE strains used acetate or oxaloacetate white 50% of the I strains used acetate and none was able to grow on oxaloacetate. No correlation was found between the symbiotic effectiveness of the strains and their ATP content, when grown on mannitol. The highest ATP content (9.21 nM×g protein^–1) was found in the I strain S₂₀ and the lowest (0.69 nM×g protein^–1 was found in the effective strain S₈. Numerical analysis of the patterns of utilization of the TCA cycle intermediates and related compounds indicated that the 49 strains tested formed 11 distinct groups at 86% similarity, according to Jaccard's coefficient. Several strains showed unique patterns of utilization and can be clearly identified under laboratory conditions.Contribution no.225 Station de Recherches, Agriculture Canada.     

Piperine,an active ingredient of white pepper,suppresses the growth of multidrug-resistant toxigenic Vibrio cholerae and other pathogenic bacteria

Emergence and rapid spread of multidrug-resistant (MDR) bacteria including Vibrio cholerae are a global public health issue. Much attention has been paid to natural compounds, such as spices and herbs to find novel antimicrobial compounds as they are considered to be cheaper alternatives to develop as a drug. Here, we show that methanol extract of white pepper could inhibit the growth of V. cholerae O1 El Tor variant, responsible for the recent outbreaks/epidemics. Furthermore, we demonstrate for the first time that piperine, the major component of white pepper, showed a dose-dependent bactericidal effect on V. cholerae growth irrespective of their biotypes and serogroups in the presence of 200 and 300 µg ml⁻¹ of piperine, respectively. Piperine also inhibited the growth of MDR strains of Pseudomonas aeruginosa, Escherichia coli isolated from poultry and enterohemorrhagic/enteroaggregative E. coli O104 in the presence of 200 µg ml⁻¹. Interestingly, we did not observe any significant inhibitory effect of piperine on E. coli strains isolated from healthy person even up to 200 µg ml⁻¹. Our data suggest that piperine could be a novel antimicrobial agent in therapeutic and preventive applications against infections caused by pathogenic bacteria including MDR strains.     

Selective medium for primary isolation of members of the tribeProteeae

A selectiveProteeae medium (SPM) for isolation and preliminary detection of species of generaProteus, Morganella, andProvidencia was evaluated. The SPM contains tryptose phosphate agar with phenolphthalein monophosphate (as substrate for phosphatase activity), bile salts and polymyxin B (as inhibitors). The selectivity of the SPM was tested by the ecometric method of quality assurance of culture media. Fourteen reference cultures of enterobacteria and fifty-four strains ofProteeae were tested for their absolute growth index (AGI). Ninety-five percent of testedProteeae strains display an AGI above 2.5. The detected phosphatase activity proved to be able to discriminate colonies of members of the tribeProteeae. The ability of SPM for primary isolation of members ofProteeae was tested on food and clinical material and 94 strains were isolated. In addition, the SPM was employed in routine practice of clinical microbiology. From 1016 clinical samples (stool, urine, vaginal and urethral swabs), 57 strains ofProteeae were detected by the SPM in contrast to 35 strains by the routine procedure. The difference amounts to nearly 40%.