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PubMed1983

Hydroxystreptomycin production and resistance in Streptomyces glaucescens.

The wild-type strain of Streptomyces glaucescens produces hydroxystreptomycin and shows an inherent natural resistance to streptomycin group aminoglycoside antibiotics. Cell free extracts of the wild-type strain were able to inactivate streptomycin, hydroxystreptomycin and dihydrostreptomycin in the presence of ATP. The phosphotransferase did not inactivate other aminoglycoside antibiotics, including spectinomycin. Mutant strains were isolated, which were highly sensitive to streptomycin group aminoglycosides, had no measurable phosphotransferase activity and were unable to form detectable amounts of hydroxystreptomycin. This suggests either a correlation between phosphotransferase activity, streptomycin resistance and hydroxystreptomycin formation or defects in more than one gene in the strS mutant strains tested.

Anti-Bacterial Agents↗
PubMed2002

Cloning and expression of the phosphotriesterase gene hocA from Pseudomonas monteilii C11.

The cloning of a gene encoding the novel phosphotriesterase from Pseudomonas monteilii C11, which enabled it to use the organophosphate (OP) coroxon as its sole phosphorus source, is described. The gene, called hocA (hydrolysis of coroxon) consists of 501 bp and encodes a protein of 19 kDa. This protein had no sequence similarity to any proteins in the SWISS-PROT/GenBank databases. When a spectinomycin-resistance cassette was placed in this gene, phosphotriesterase activity was abolished and P. monteilii C11 could no longer grow with coroxon as the sole phosphorus source. Overexpression and purification of HocA as a maltose-binding protein fusion produced a protein having a broad substrate specificity across oxon and thion OPs. Michaelis-Menten kinetics were observed with the oxon OPs, but not with the thion OPs. End-product inhibition was observed for coroxon-hydrolytic activity. Increased expression of hocA was observed from an integrative hocA-lacZ fusion when cultures were grown in the absence of phosphate, suggesting that it might be part of the Pho regulon, but the phosphate-regulated promoter was not cloned in this study. This is believed to be the first study in which a gene required for an organism to grow with OP pesticides as a phosphorus source has been isolated.

Aryldialkylphosphatase↗
PubMed1976

Isolation and characterization of mutants of Escherichia coli K12 resistant to the new aminoglycoside antibiotic, amikacin.

Spontaneous mutants of Escherichia coli K12 that are resistant to the new aminoglycoside antibiotic, amikacin, were isolated. These mutants have simultaneously acquired cross-resistance to kanamycin, gentamicin and neomycin but not to streptomycin or spectinomycin. Sensitivity of the mutant strains to the non-aminoglycoside antibiotics, ampicillin, tetracycline and polymyxin, was unaffected. The mutation responsible for amikacin resistance was mapped by PI transduction and found to be tightly linked to strA, distal with respect to spcA and aroE.

Amikacin↗
PubMed1985

PSE-4 beta lactamase: a serotype-specific enzyme in Pseudomonas aeruginosa.

PSE-4 enzyme is the most common plasmidic beta lactamase in Pseudomonas aeruginosa but its production is invariably non-transferable by conjugation. Of 20 PSE-4+ isolates from 10 separate sources, 16 were serotype O:16 and two belonged to the related O:2(b) serotype. One of the two remaining organisms was not O-typable and the other was agglutinated by several unrelated antisera. Examination of additional O:16 strains confirmed the unusual frequency of PSE-4 enzyme in this serotype. None of the PSE-4+ strains was able to transfer carbenicillin resistance in mating experiments and none contained extrachromosomal DNA. Two explanations of the relationship between enzyme production and O antigenicity are proposed. PSE-4 production may be transmissible, perhaps by transduction, between strains of O:16 or related serotypes, or PSE-4+ P. aeruginosa may represent a disseminated subtype. A third hypothesis, that the PSE-4 coding element carried serotype determinants, was discounted. PSE-4+ and PSE-4- P. aeruginosa strains of O:16 and related serotypes were found to represent a definite cluster by their phage-susceptibility pattern and pyocin type (type 1). The only characters linked to PSE-4 production were resistance to spectinomycin, streptomycin and sulphonamide and the genes responsible for these characters seemed to occur on the PSE-4 coding element.

Anti-Bacterial Agents↗
PubMed1985

Susceptibility to antimicrobial agents and analysis of plasmids in gentamicin- and methicillin-resistant Staphylococcus aureus from Dublin hospitals.

Methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) strains isolated from Dublin Hospitals were classified into two groups (phenotypes). Phenotype-I strains expressed high level resistance to gentamicin and were susceptible to fusidic acid; strains resistant to tetracycline harboured a 3 X 10(6)-mol. wt plasmid. Strains in phenotype II usually expressed low level resistance to gentamicin, were resistant to fusidic acid and often harboured a (22-24) X 10(6)-mol. wt plasmid that specified resistance to ethidium bromide, tetracycline, kanamycin, neomycin and trimethoprim, or to combinations of these markers. A few phenotype-II strains expressed higher levels of resistance to gentamicin and other aminoglycosides. All MGRSA strains carried a 21 X 10(6)-mol. wt plasmid conferring resistance to penicillin, ethidium bromide, cadmium and mercury. Gentamicin resistance was invariably chromosomal and all strains carried chromosomal resistance to methicillin, erythromycin, streptomycin and spectinomycin. Several methicillin-resistant S. aureus (MRSA) strains isolated before the emergence of gentamicin resistance harboured a 21 X 10(6)-mol. wt penicillinase plasmid with the same restriction endonuclease profile as that from some MGRSA strains. Some MRSA strains carried other plasmids related to those found in MGRSA strains.

Anti-Bacterial Agents↗
PubMed1986

Acquisition of a gene encoding mannose-resistant haemagglutinating fimbriae by a resistance plasmid during long-term urinary infection.

Urine was collected twice weekly from one patient during a 25-month period. Escherichia coli harbouring a resistance plasmid, pUK28, which confers trimethoprim, ampicillin, sulphamethoxazole, spectinomycin and streptomycin resistance was identified in the urine. Carriage of strains containing plasmid pUK28 was observed during three separate periods which totalled 16 months, even though the patient did not receive antibacterial drug therapy for most of that time. The plasmid was able to acquire the genes responsible for mannose-resistant haemagglutination and these genes were increasingly associated with the plasmid towards the end of the study period.

Aged↗
PubMed1991

Distribution and transferability of plasmids encoding trimethoprim resistance in urinary pathogens from Greece.

Of 505 strains of Enterobacteriaceae responsible for significant bacteriuria and isolated from hospital patients in two Greek cities in 1989, 151 strains (30%) were resistant to trimethoprim (MIC greater than or equal to 4 mg/L) and 220 (44%) were resistant to sulphamethoxazole (MIC greater than or equal to 64 mg/L); 127 (84%) of the trimethoprim-resistant strains exhibited high-level resistance (MIC greater than 1024 mg/L) and 121 (80%) were additionally resistant to four or more other antibiotics. Plasmids were detected in 141 (93%) of the trimethoprim-resistant strains. Trimethoprim resistance was encoded on self-transmissible plasmids in 79 (52%) of the resistant strains, and in a further seven strains (5%), plasmids coding for trimethoprim resistance could be mobilised by X+ factor. Co-transfer of various other antimicrobial resistances with trimethoprim resistance was observed, tetracycline resistance being the most common. The low degree of linkage observed between trimethoprim resistance and resistance to streptomycin and spectinomycin suggests that Tn7 is relatively uncommon in Greece. Classification of trimethoprim-resistance plasmids on the basis of their antimicrobial-resistance patterns and molecular mass revealed 39 different profiles. Overall, these findings differ from those from other European countries where the prevalence of transferable high-level trimethoprim resistance is low and where chromosomal Tn7-encoded trimethoprim resistance is common.

Bacteriuria↗
PubMed1995

Utility of ribotyping, restriction endonuclease analysis and pulsed-field gel electrophoresis to discriminate between isolates of Neisseria gonorrhoeae of serovar IA-2 which require arginine, hypoxanthine or uracil for growth.

Neisseria gonorrhoeae isolates that require arginine--i.e., either citrulline (C), or ornithine (O)--uracil (U) and hypoxanthine (H) have generally been considered to be similar when characterised by auxotype, serovar and plasmid content. The MICs of penicillin, tetracycline, erythromycin, spectinomycin, cefoxitin and ceftriaxone for 552 isolates belonging to serovar IA-2 with these phenotypes were found to be similar. Therefore, restriction fragment length polymorphism analysis of rRNA genes (ribotyping), restriction enzyme (RE) analysis of chromosomal DNA, and pulsed-field gel electrophoresis (PFGE) were evaluated to determine whether these isolates could be distinguished by molecular methods. A subset of 27 isolates of N. gonorrhoeae that were OUH-requiring, CUH-requiring or OH-requiring, belonged to serovar IA-2 and carried a 2.6-MDa plasmid, were selected for further study. Based on the RE analysis of SmaI-digested genomic DNA, the 27 isolates fell into a single RE pattern, five ribotypes and 17 PFGE profiles which did not correlate with the specific arginine-requiring subtypes of these isolates. Each ribotype varied by the presence of only a single fragment, which was of a different size in each pattern, and 17 (63%) of the 27 isolates belonged to ribotype I. PFGE yielded the highest level of discrimination with 17 different profiles.

Anti-Bacterial Agents↗
PubMed2004

Phenylobacterium lituiforme sp. nov., a moderately thermophilic bacterium from a subsurface aquifer, and emended description of the genus Phenylobacterium.

A facultative anaerobic bacterium, strain FaiI3(T), was isolated from samples collected from the free-flowing waters of a bore well (Fairlea Bore, registration number 3768) which taps into the Australian Great Artesian Basin subsurface thermal aquifer. Strain FaiI3(T) developed yellow to pale-yellow colonies (0.5-1.5 mm) after 48 h. The non-spore forming rods (0.5x1-3 microm) were slightly curved, occurred singly and as pairs and were motile with a single polar flagellum. Cells tended to form clumps in liquid medium and rosettes were commonly observed. The cells stained Gram-negative and electron micrographs of thin sections revealed a multi-layered complex Gram-negative cell wall structure. Strain FaiI3(T) grew optimally at 40-41 degrees C, with growth observed at 45 degrees C but not at 50 degrees C. The pH growth range was between pH 6 and 9 and optimal growth occurred between pH 6 and 6.5. Strain FaiI3(T) grew best with yeast extract as the sole carbon and energy source. Peptone, yeast extract, acetate, xylose, sucrose, glucose, glycerol, succinate, butyrate, lactate, fumarate, citrate, L-phenylalanine, cellobiose and gelatin supported growth but maltose, fructose, glycine, ethanol, benzoate and oxalate did not. Tyrosine was produced from L-phenylalanine. Strain FaiI3(T) was catalase-positive and oxidase-negative and did not hydrolyse starch. Growth was inhibited by neomycin, tetracycline, streptomycin, chloramphenicol, ampicillin, vancomycin and spectinomycin. The G+C content was determined to be 66.5+/-0.5 mol%. On the basis of the 16S rRNA gene sequence analysis, strain FaiI3(T) was assigned as a novel species of the genus Phenylobacterium, Phenylobacterium lituiforme sp. nov. in the order Caulobacterales, subclass alpha-Proteobacteria, class Proteobacteria. The type strain is FaiI3(T) (=ATCC BAA-294(T)=DSM 14363(T)).

Anaerobiosis↗
PubMed2005

Methanomethylovorans thermophila sp. nov., a thermophilic, methylotrophic methanogen from an anaerobic reactor fed with methanol.

A novel thermophilic, obligately methylotrophic, methanogenic archaeon, strain L2FAW(T), was isolated from a thermophilic laboratory-scale upflow anaerobic sludge blanket reactor fed with methanol as the carbon and energy source. Cells of strain L2FAW(T) were non-motile, irregular cocci, 0.7-1.5 mum in diameter and usually occurred singly (sometimes forming clusters of two or four cells). The cells stained Gram-negative and lysed immediately in 0.1 % (w/v) SDS. Growth was inhibited by chloramphenicol and tetracycline, but not by penicillin, bacitracin, spectinomycin, vancomycin or kanamycin. Methanol and mono-, di- and trimethylamine were used as substrates, but H2/CO2, formate, acetate, propanol, dimethyl sulfide and methanethiol were not. The temperature range for growth was 42-58 degrees C, with an optimum at 50 degrees C. The fastest growth was observed at a salinity below 100 mM NaCl; no growth occurred above 300 mM NaCl. The optimal pH for growth was 6.5; growth was observed from pH 5 to pH 7.5. The G+C content of the genomic DNA was 37.6 mol%. Analysis of the 16S rRNA gene sequence and the partial methyl-CoM reductase gene sequence revealed that the organism was phylogenetically closely related to Methanomethylovorans hollandica DMS1T (98 % similarity for the 16S rRNA gene sequence and 91 % similarity for the methyl-CoM reductase gene sequence). The DNA-DNA relatedness between L2FAW(T) and Methanomethylovorans hollandica DMS1T was 46 %. On the basis of these results, strain L2FAW(T) (=DSM 17232T=ATCC BAA-1173T) represents the type strain of a novel species, for which the name Methanomethylovorans thermophila sp. nov. is proposed.

Anaerobiosis↗
PubMed2006

Distribution and characterization of integrons in various serogroups of Vibrio cholerae strains isolated from diarrhoeal patients between 1992 and 2000 in Kolkata, India.

A total of 133 clinical strains of Vibrio cholerae comprising 44 strains of O1, 45 strains of O139 and 44 strains of non-O1, non-O139 serogroups isolated from hospitalized patients in Kolkata, India, from 1992 to 2000 was examined for the presence of class 1, 2 and 4 integrons. By PCR and DNA sequencing, seven strains of O1, one strain of O139 and six strains of non-O1, non-O139 serogroups were found to contain class 1 integron-harbouring genes aadA1, aadA2 (encoding resistance to streptomycin and spectinomycin), blaP1 (resistance to beta-lactams), aar-3 (resistance to rifampicin), aacA4 (resistance to kanamycin and gentamicin), and dfrA1 and dfrA15 (resistance to trimethoprim). Most strains produced one or two bands using primers specific for the amplification of the variable region where the antibiotic-resistance genes are located, and their sizes ranged from 700 to 1250 bp. However, one strain of V. cholerae O1 isolated in 1994 gave a 2483 bp fragment, the largest fragment so far found in a class 1 integron of V. cholerae O1. No strain was positive for the intI2 gene. All V. cholerae strains, regardless of serogroup, were positive for the intI4 gene by PCR and using a colony hybridization test. Amplification of the intI4 gene by PCR yielded a 2200 bp fragment (1260 bp larger than the expected size) from three V. cholerae O139 strains isolated in 1999. Sequence analysis of this amplicon revealed an insertion of IS1359 in the middle of the intI4 gene. These data indicate that a class 1 integron is present in some clinical strains of V. cholerae isolated in Kolkata, India, and that a class 4 integron is ubiquitously distributed among V. cholerae strains regardless of serogroup.

Anti-Bacterial Agents↗
PubMed2006

Genetic characterization of multidrug resistance in Shigella spp. from Japan.

This study characterized the genetic basis of antimicrobial resistance of a number of Shigella spp. isolated from humans from 2000 to 2004 in Hiroshima prefecture, Japan. A total of 26 isolates of Shigella spp. were included in this study. Antimicrobial susceptibility tests revealed high levels of resistance, especially to ampicillin, streptomycin, trimethoprim, tetracycline, nalidixic acid and ciprofloxacin. PCR and DNA sequencing were used for screening and characterization of antibiotic-resistance determinants. PCR sequencing analysis revealed the presence of only one type of class 1 integron in one isolate of Shigella sonnei. This class 1 integron was 1904 bp and contained two gene cassettes: a probable esterase/lipase (estX) and aadA1, which confers resistance to streptomycin and spectinomycin. Two types of class 2 integron were identified in this study. One was the classic type (2158 bp) and carried the three conserved resistance gene cassettes of the class 2 integron, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively. This type was detected in both Shigella sonnei (14 isolates) and Shigella flexneri (five isolates). The other type was shorter (1313 bp) and carried only two gene cassettes, dfrA1 and sat1. This integron was detected in a single isolate of Shigella sonnei. PFGE patterns showed limited diversity within clusters of the same species. Furthermore, an extended-spectrum beta-lactamase gene, bla(OXA-30), which confers resistance to ampicillin, was characterized in all isolates of Shigella flexneri except the oldest strain, which was isolated in 2000. Southern blot hybridization and conjugation experiments showed that bla(OXA-30) was located in the chromosome.

Anti-Infective Agents↗
PubMed2003

Construction and complementation of the first auxotrophic mutant in the spirochaete Leptospira meyeri.

In bacteria, the first reaction of the tryptophan biosynthetic pathway involves the conversion of chorismate and glutamine to anthranilate by the action of anthranilate synthase, which is composed of the alpha (trpE gene product) and beta (trpG gene product) subunits. In this study, the tryptophan biosynthetic gene trpE of the spirochaete Leptospira meyeri was interrupted by a kanamycin-resistance cassette by homologous recombination. The trpE double cross-over mutant was not able to grow on solid or in liquid EMJH medium. In contrast, the trpE mutant showed a wild-type phenotype when tryptophan or anthranilate was added to the media, therefore showing that disruption of the L. meyeri trpE gene resulted in tryptophan auxotrophy. The authors have also characterized a second selectable marker that allows the construction of a spectinomycin-resistant L. meyeri-E. coli shuttle vector and the functional complementation of the L. meyeri trpE mutant.

Anthranilate Synthase↗
PubMed1998

A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo.

Splicing of the group I intron of the T4 thymidylate synthase (td) gene was uncoupled from translation by introducing stop codons in the upstream exon. This resulted in severe splicing deficiency in vivo. Overexpression of a UGA suppressor tRNA partially rescued splicing, suggesting that this in vitro self-splicing intron requires translation for splicing in vivo. Inhibition of translation by the antibiotics chloramphenicol and spectinomycin also resulted in splicing deficiency. Ribosomal protein S12, a protein with RNA chaperone activity, and CYT-18, a protein that stabilizes the three-dimensional structure of group I introns, efficiently rescued the stop codon mutants. We identified a region in the upstream exon that interferes with splicing. Point mutations in this region efficiently alleviate the effect of a nonsense codon. We infer from these results that the ribosome acts as an RNA chaperone to facilitate proper folding of the intron.

Bacteriophage T4↗
PubMed1997

Requirement for the H phosphoprotein in photosystem II of Chlamydomonas reinhardtii.

To dissect the expression of the psbB gene cluster of the Chlamydomonas reinhardtii chloroplast genome and to assess the role of the photosystem II H-phosphoprotein (PSII-H) in the biogenesis and/or stabilization of PSII, an aadA gene cassette conferring spectinomycin resistance was employed for mutagenesis. Disruption of the gene cluster has no effect on the abundance of transcripts of the upstream psbB/T locus. Likewise, interruption of psbB/T and psbH with a strong transcriptional terminator from the rbcL gene does not influence transcript accumulation. Thus, psbB/T and psbH may be independently transcribed, and the latter gene seems to have its own promoter in C. reinhardtii. In the absence of PSII-H, translation and thylakoid insertion of chloroplast PSII core proteins is unaffected, but PSII proteins do not accumulate. Because the deletion mutant also exhibits PSII deficiency when dark-grown, the effect is unrelated to photoinhibition. Turnover of proteins B and C of PSII and the polypeptides PSII protein A and PSII protein D is faster than in wild-type cells but is much slower than that observed in other PSII-deficient mutants of C. reinhardtii, suggesting a peripheral location of PSII-H in PSII. The role of PSII-H on PSII assembly was examined by sucrose gradient fractionation of pulse-labeled thylakoids; the accumulation of high-molecular-weight forms of PSII is severely impaired in the psbH deletion mutant. Thus, a primary role of PSII-H may be to facilitate PSII assembly/stability through dimerization. PSII-H phosphorylation, which possibly occurs at two sites, may also be germane to its role in regulating PSII structure, stabilization, or activity.

Animals↗
PubMed2001

Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Return of a gene to its pre-endosymbiotic origin.

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS alpha-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion at ndhF-trnL only expressed one size of the ASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS alpha-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels in leaves and total Trp levels in seeds. Resistance to an AS inhibitor, 5-methyl-Trp, was found during seed germination and in suspension cultures of the transplastomic plants. The resistance to the selection agent spectinomycin and to 5-methyl-Trp was transmitted maternally. These results demonstrate the feasibility of modifying the biosynthetic pathways of important metabolites through transformation of the plastid genome by relocating a native gene from the nucleus to the plastid genome. Very high and uniform levels of gene expression can be observed in different lines, probably due to the identical insertion sites, in contrast to nuclear transformation where random insertions occur.

Anthranilate Synthase↗
PubMed1979

Ribosome-thylakoid association in peas: influence of anoxia.

Isolated pea chloroplast thylakoids ordinarily have ribosomes attached which survive sequential washes. Extensive in vivo loss of these thylakoidbound ribosomes occurred if the pea plants were placed in the dark without O(2) for 2 or more hours. This loss was indicated from measurements of both the total thylakoid-bound RNA levels, and the capacity for amino acid incorporation into proteins on the addition of soluble enzymes for protein synthesis. Stroma ribosome profiles lost any indication of polysome structure due to the same anoxic treatment in vivo. The return of ribosomes to the thylakoids when plants were placed in the light in air occurred over an 8-hour time course. This return was prevented by lincomycin, spectinomycin, and chloramphenicol, indicating a requirement for protein synthesis steps in the stroma at some point in the reassociation process.

Journal Article↗
PubMed1982

Induction of HCO(3) Transporting Capability and High Photosynthetic Affinity to Inorganic Carbon by Low Concentration of CO(2) in Anabaena variabilis.

The apparent affinity of photosynthesis for inorganic carbon in Anabaena variabilis strain M-3 increased during the course of adaptation from high to low CO(2) concentration (5% and 0.03% v/v CO(2) in air, respectively). This was attributed to an increased ability of the cells to accumulate inorganic carbon during the course of adaptation to low CO(2) conditions. The release of phycobiliproteins was used to evaluate the sensitivity of the cells to lysozyme treatment followed by osmotic shock. High CO(2)-grown cells were more sensitive to this treatment than were low CO(2) ones. The efflux of inorganic carbon from cells preloaded with radioactive bicarbonate is faster in high than it is in low CO(2)-adapted cells. It is postulated that the cell wall or membrane components undergo changes during the course of adaptation to low CO(2) conditions. This is supported by electron micrographs showing differences in the cell wall appearance between high and low CO(2)-grown cells. The increasing ability to accumulate HCO(3) (-) and the lessened sensitivity to lysozyme during adaptation to low CO(2) conditions depends on protein synthesis. The increase in affinity for inorganic carbon during the adaptation to low CO(2) conditions is severely inhibited by the presence of spectinomycin. Incubation in the light significantly lessens the time required for the adaptation to low CO(2) conditions.

Journal Article↗