Sporulation of a bacitracin-sensitive mutant of Bacillus licheniformis is self-inhibited by bacitracin

A mutant of Bacillus licheniformis (BLU166) sensitive to its own antibiotic bacitracin was isolated and the mutation bcr-l was mapped close to the bacitracin synthetase genes. The sensitivity was shown to be specific for bacitracin. Two further bacitracin-sensitive strains were constructed, one (BLU171) with normal ability to synthesize bacitracin, and one (BLU170) a bacitracin non-producer. In addition to an increased sensitivity of growing cells to bacitracin, sporulation of the mutant strain BLU171 was self-inhibited by bacitracin. It is concluded that (1) there might exist at least two levels of resistance to bacitracin; (2) mutation bcr-1 affects a 'structural' component, which may protect the sensitive reaction of cell-wall biosynthesis; (3) sporulation is affected to a greater extent by bacitracin than vegetative growth; and (4) synthesis of bacitracin is independent of the presence of this resistance mechanism since the sensitive mutant produces similar amounts of the antibiotic to the wild-type strain.     

Carbomycin resistance in mouse L cells

A mutant has been isolated from the mouse cell line LM(TK-) which is stably resistant to the macrolide antibiotic, carbomycin. Mitochondrial protein synthesis in this mutant was carbomycin resistant and chloramphenicol sensitive. Fusions between carbomycin-resistant and -sensitive cells produced hybrids, most of which were sensitive to 10 microgram/ml carbomycin. At 7.5 microgram carbomycin/ml, the average population resistance is low initially but increases with time. Carbomycin-resistant cells were enucleated and fused with carbomycin-sensitive cells under a variety of selective regimes designed to allow growth of carbomycin-resistant cytoplasmic hybrids (cybrids). No transfer of carbomycin resistance via the cytoplasm was detected. Karyoplasts from carbomycin-resistant cells showed a low transfer of resistance to 7.5 microgram carbomycin/ml in karyoplast-cell fusions. Carbomycin resistance in this mutant is therefore most likely encoded in a nuclear gene.     

Isolated cell behavior drives the evolution of antibiotic resistance

Bacterial antibiotic resistance is typically quantified by the minimum inhibitory concentration (MIC), which is defined as the minimal concentration of antibiotic that inhibits bacterial growth starting from a standard cell density. However, when antibiotic resistance is mediated by degradation, the collective inactivation of antibiotic by the bacterial population can cause the measured MIC to depend strongly on the initial cell density. In cases where this inoculum effect is strong, the relationship between MIC and bacterial fitness in the antibiotic is not well defined. Here, we demonstrate that the resistance of a single, isolated cell—which we call the single‐cell MIC (scMIC)—provides a superior metric for quantifying antibiotic resistance. Unlike the MIC, we find that the scMIC predicts the direction of selection and also specifies the antibiotic concentration at which selection begins to favor new mutants. Understanding the cooperative nature of bacterial growth in antibiotics is therefore essential in predicting the evolution of antibiotic resistance.     

Comparative analysis of a biofilm-forming Staphylococcus epidermidis strain and its adhesion-positive, accumulation-negative mutant M7

Abstract We have isolated a stable slime-negative mutant, M7, from the wild-type Staphylococcus epidermidis RP62A by mitomycin mutagenesis. Besides its inability to produce slime in the test tube this mutant differed also in two other properties from its parent strain: it lacked the ability to accumulate on a surface, and it did not produce a 115 kDa and a 18 kDa extracellular protein. In all other tested properties such as initial adherence, growth rate, cell-wall composition, surface characteristics, DNA restriction profile, the presence of a 29 kb antibiotic resistance plasmid, and antimicrobial susceptibility profile, M7 was indistinguishable from its wild-type. The mutant is an important basis for further study of the pathogenesis of polymer-associated S. epidermidis infections.     

Increase of methicillin resistance in Staphylococcus aureus caused by deletion of a gene whose product is homologous to lytic enzymes.

A spontaneous high-level methicillin-resistant mutant, SRM1648, for which the MIC of methicillin is 1,600 microg/ml, was isolated on a plate containing 400 microg of the antibiotic/ml on which had been cultured the low-level methicillin-resistant Staphylococcus aureus SR17238, for which the MIC is 6.3 microg/ml. Analysis of the chromosomal DNAs of the mutant and the parental strains by the restriction landmark genomic scanning method with two-dimensional electrophoresis of restriction fragments revealed a 1.6-kb deletion in the chromosome of the mutant. The HindIII fragment of 2.5 kb containing this deleted region was cloned into a plasmid vector and introduced into the parental strain. A deletion mutant reconstructed in the presence of a low concentration of methicillin by integration and excision of the recombinant plasmid exhibited a high level of resistance (methicillin MIC, 1,600 microg/ml), confirming that the deletion had caused the elevation of the resistance level. Sequence analysis indicated that the deletion occurred in three consecutive open reading frames (ORFs). The predicted amino acid sequence of the first ORF showed high homology with both RelA and SpoT of Escherichia coli, which are involved in the synthesis and hydrolysis of guanosine 5',3'-polyphosphate, and that of the third ORF showed a relatively high homology to the lytic enzyme encoded by the lytC gene of Bacillus subtilis. We also isolated another high-level resistant mutant with a deletion within the third ORF, which suggested that inactivation of some lytic enzyme resulted in the increased resistance.     

Comparison of the growth promoting activities and toxicities of various auxin analogs on cells derived from wild type and a nonrooting mutant of tobacco

A naphthaleneacetic acid tolerant mutant isolated from a mutagenized culture of tobacco mesophyll protoplasts and impaired in root morphogenesis has been previously characterized by genetic analysis. To understand the biochemical basis for naphthaleneacetic acid resistance, cells derived from this mutant and from wild-type tobacco were compared for their ability to respond to various growth regulators. The growth promoting abilities and cytotoxicities of auxin analogs were different for mutant and wild-type cells. These different activities were not correlated with increased rate of conjugation or breakdown of the auxins by mutant cells. These observations, as well as previous studies on the interaction of the mutant with Agrobacterium, suggest that mutant resistance to auxins is not a result of a specific modification of the process by which auxins induce cell killing, but to a more general alteration of the cellular response to auxin. A screening of auxin-related molecules which induce cell death in wild-type cells but not mutant cells without promoting growth in either was performed. p-Bromophenyleacetic acid was found to display these characteristics.     

Cytoplasmic membrane proteins of spectinomycin-susceptible and -resistant strains of Neisseria gonorrhoeae.

Cytoplasmic membranes were isolated and examined from two spectinomycin-susceptible and three spectinomycin-resistant clinical strains of Neisseria gonorrhoeae. A laboratory-derived spectinomycin-resistant mutant, obtained by serial passage on gradually increasing concentrations of the antibiotic, and a susceptible revertant, spontaneously arising from one of the resistant clinical strains, were also studied. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis revealed that a major protein, comprising about 7% of total cytoplasmic membrane protein (molecular weight 24,000), was absent in the three clinically isolated spectinomycin-resistant strains. In a revertant, this protein reappeared. During treatment of one of the susceptible strains with spectinomycin, the protein disappeared. However, this correlation was not maintained in the laboratory-derived spectinomycin-resistant mutant. This mutant was of comparable resistant to the clinical isolates, but the 24,000-molecular-weight protein was present in normal quantities. In addition, spectinomycin resistant in clinical isolates was variable compared with stable resistance exhibited by the laboratory-derived mutant. These findings suggested that differences in laboratory-derived versus clinical spectinomycin resistance may be due to different types of resistance mutations.     

Isolation and characterization of mevinolin resistant mutants of Saccharomyces cerevisiae

Mutant of Saccharomyces cerevisiae resistant to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase (EC1.1.1.34) were isolated and one mutant (MV71) was extensively characterized. While growth of resistant strains in the presence of mevinolin was growth. Diploids produced by mutant/wild-type matings showed levels of mevinolin resistance which indicated incomplete dominance. Sterol synthesis in the presence of mevinolin was inhibited in strain MV71 but to a lesser degree than seen in the wild-type strain. All mevinolin resistant mutants also demonstrated a slight resistance to the antibiotic nystatin. The subcellular location of HMGCoA reductase activity in MV71 and the wild-type strain were determined and it was shown that yeast HMGCoA reductase is not regulated by a dephosphorylation mechanism as has been shown for mammalian reductases. In vivo and in vitro studies of strain MV71 and the wild-type indicated that mevinolin resistance did not result in changes in HNGCoA reductase activity as has been demonstrated in mammalian systems. Based on growth data, sterol analysis, and the lack of detection of HMGCoA reductase activity differences between strain MV71 and the wild-type, mevinolin resistance is concluded to result possibly from a mutation in HMG2, one of the two functional yeast HMGCoA reductase genes, which accounts for a minor (up to 17%) amount of total cellular reductase activity.     

Biodegradation of Phosphonomycin by Rhizobium huakuii PMY1

The biodegradation by Rhizobium huakuii PMY1 of up to 10 mM phosphonomycin as a carbon, energy, and phosphorus source with accompanying P_i release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by Rhizobium spp.     

Characterization of a respiratory mutant of Escherichia coli with reduced uptake of aminoglycoside antibiotics

A strain of Escherichia coli (NSW77) which is partially resistant to streptomycin was isolated by selecting for growth on plates supplemented with 12.5 μg/ml streptomycin, a concentration which completely inhibits growth of wild-type strains. The low-level resistance of the mutant appears to result from a reduced ability to accumulate streptomycin intracellularly. In addition, the mutant strain is unable to use succinate for growth because of a defective respiratory chain. Thus, membranes of the mutant strain were found to have approximately half the NADH and D-lactate oxidase activity of the parent strain. Succinate oxidase activity was reduced more drastically, to a level of 7% that of the parent strain. Moreover, membranes of the mutant were found to contain demethyl-menaquinone and, in place of ubiquinone, a structural analogue, 2-octaprenyl-3-methyl-6-methoxy-1,4 benzoquinone. The mutation responsible for both the Suc⁻ phenotype and partial resistance to streptomycin was found to be located near minute 15 on the bacterial chromosome. Both the biochemical and genetic evidence suggests that the mutation in strain NSW77 resides in the ubi F gene. Another previously characterized ubi F strain was also found to have a reduced capacity to take up an aminoglycoside antibiotic (gentamicin). These results suggest that the respiratory defects in ubi F strains are responsible for the reduced capacity of such strains to accumulate aminoglycosides.     

Extrachromosomal inheritance of paromomycin resistance in Saccharomyces cerevisiae. Genetic and biochemical characterization of mutants

Summary In Saccharomyces cerevisiae, mutants were isolated which show high resistance to the aminoglycoside paromomycin. Amino acid incorporation of mitochondria isolated from such mutant strains proved also to be paromomycin resistant. All of them are cross-resistant to the structurally related antibiotic neomycin. Three independent methods revealed the resistance to be extrachromosomally, presumably mitochondrially inherited.     

Role for tandem duplication and lon protease in AcrAB-TolC- dependent multiple antibiotic resistance (Mar) in an Escherichia coli mutant without mutations in marRAB or acrRAB

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.     

Antibiotic resistant bacteria in Windermere and two remote upland tarns in the English Lake District

The incidence of antibiotic resistance was determined in over 2000 bacteria which were divided into the following groups: faecal streptococci, coliforms (excluding Escherichia coli), E. coli, Pseudomonas spp. and aquatic bacteria (i.e. bacteria predominant in the lake water which were excluded from the previous four categories). The isolates were obtained from the water of Windermere (English Lake District) and from a sewage effluent which entered the lake. With the exception of the faecal streptococci, the incidence of antibiotic resistance was higher in the bacteria isolated from the lake water than in those from the effluent, and ranked according to groups Pseudomonas spp. greater than E. coli greater than aquatic bacteria greater than coliforms greater than faecal streptococci. The highest incidence of multiple resistance was found among the pseudomonads. When corrected for the relative size of each population the pool of antibiotic resistance in the aquatic bacteria was by far the largest. The incidence of antibiotic resistance in aquatic bacteria isolated from Windermere was, however, lower than in those isolated from two remote upland tarns. This finding may have been due to differences in the species composition of the three sites except that the same results were obtained when only fluorescent pseudomonads were tested. The upland tarns were not totally isolated from man and other animals but did not receive any sewage or other effluents and therefore the results were surprising. Possible explanations include a lack of susceptibility in aquatic bacteria and increased resistance associated with growth in nutrient poor environments.     

Improvement of antibiotic titers from Streptomyces bacteria by interactive continuous selection

We have applied a technique of interactive continuous selection (ICS) to the isolation of streptomycin-resistant mutants of the streptomycin-producing organism, Streptomyces griseus. A series of mutants, each with a different colonial morphology and expressing successively greater resistance to streptomycin, was isolated during the course of selection. Takeover of the mutants has been correlated with changes in on-line estimates of streptomycin concentration such that these estimates may be used as a real-time measure of the genetic state of the cell population. When grown in the medium employed for ICS, mutants expressed increased antibiotic production titers; the best mutant produced 10 to 20 times more streptomycin than the parent strain. Absolute improvements in the maximum specific growth rate and intrinsic resistance to streptomycin did not account for the observed growth advantage of all mutants. Rather, each mutant exhibited relative increases in specific growth rate at increasing concentrations of streptomycin. (c) 1996 John Wiley & Sons, Inc.     

Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.     

A phosphonate-induced gene which promotes Penicillium-mediated bioconversion of cis-propenylphosphonic acid to fosfomycin

Penicillium decumbens is able to epoxidize cis-propenylphosphonic acid (cPA) to produce the antibiotic fosfomycin [FOM; also referred to as phosphonomycin and (-)-cis-1,2-epoxypropylphosphonic acid], a bioconversion of considerable commercial significance. We sought to improve the efficiency of the process by overexpression of the genes involved. A conventional approach of isolating the presumed epoxidase and its corresponding gene was not possible since cPA epoxidation could not be achieved with protein extracts. As an alternative approach, proteins induced by cPA were detected by two-dimensional gel electrophoresis. The observation that a 31-kDa protein (EpoA) was both cPA induced and overaccumulated in a strain which more efficiently converted cPA suggested that it might take part in the bioconversion. EpoA was purified, its amino acid sequence was partially determined, and the corresponding gene was isolated from cosmid and cDNA libraries with oligonucleotide probes. The DNA sequence for this gene (epoA) contained two introns and an open reading frame encoding a peptide of 277 amino acids having some similarity to oxygenases. When the gene was subcloned into P. decumbens, a fourfold increase in epoxidation activity was achieved. epoA-disruption mutants which were obtained by homologous recombination could not convert cPA to FOM. To investigate the regulation of the epoA promoter, the bialaphos resistance gene (bar, encoding phosphinothricin acetyltransferase) was used to replace the epoA-coding region. In P. decumbens, expression of the bar reporter gene was induced by cPA, FOM, and phosphorous acid but not by phosphoric acid.     

Antimicrobial susceptibility of enterococci recovered from commercial swine carcasses: effect of feed additives

Enterococcus faecium is an important nosocomial pathogen often displaying multiple antibiotic resistance. The increase in clinical isolates can be attributed in part to hospital practices in antibiotic usage, but there is concern that antibiotic-resistant strains might also originate in animals fed rations containing antibiotic growth promoters. Ingestion of meat from carcasses contaminated with faecal enterococci might then result in human colonization or resistance gene transfer to human enterococci. Because there are few comparisons of bacteria isolated from matched animals that have, or have not, been fed a diet containing antibiotic, two such groups of pig carcasses were sampled at a commercial abattoir. Forty isolates from each group of pigs were tested for their resistance to avilamycin and tylosin. Although a modest number of pigs was examined, and the number of strains of E. faecium tested was small, there was no evidence that the feeding of a growth promoter caused selection of enterococci resistant to tylosin or avilamycin.     

Isolation and Characterization of Mutations Affecting the Transport of Hexose Phosphates in Escherichia coli

Mutants of Escherichia coli defective in the hexose phosphate transport system were isolated. Negative selection by penicillin treatment or positive selection with phosphonomycin was employed. These mutants grew normally on all carbon sources other than hexose phosphates. The map location of the mutations in 18 independently isolated mutant strains was investigated by transduction crosses. All of the mutations were found to lie in the same region of the chromosome, in the region represented by min 72 on the Taylor map. The order of the genes in this region was found to be mtl-cysE-pyrE-uhp-bgl-ilv. Revertants of some of the mutants exhibited altered regulatory control of this transport system.     

Antibiotic resistant bacteria in Windermere and two remote upland tarns in the English Lake District

The incidence of antibiotic resistance was determined in over 2000 bacteria which were divided into the following groups: faecal streptococci, coliforms (excluding Escherichia coli ), E. coli, Pseudomonas spp. and aquatic bacteria (i.e. bacteria predominant in the lake water which were excluded from the previous four categories). The isolates were obtained from the water of Windermere (English Lake District) and from a sewage effluent which entered the lake. With the exception of the faecal streptococci, the incidence of antibiotic resistance was higher in the bacteria isolated from the lake water than in those from the effluent, and ranked according to groups Pseudomonas spp. > E. coli > aquatic bacteria > coliforms > faecal streptococci. The highest incidence of multiple resistance was found among the pseudomonads. When corrected for the relative size of each population the pool of antibiotic resistance in the aquatic bacteria was by far the largest. The incidence of antibiotic resistance in aquatic bacteria isolated from Windermere was, however, lower than in those isolated from two remote upland tarns. This finding may have been due to differences in the species composition of the three sites except that the same results were obtained when only fluorescent pseudomonads were tested. The upland tarns were not totally isolated from man and other animals but did not receive any sewage or other effluents and therefore the results were surprising. Possible explanations include a lack of susceptibility in aquatic bacteria and increased resistance associated with growth in nutrient poor environments.