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1.
Antimicrob Agents Chemother ; 65(8): e0061121, 2021 07 16.
Article in English | MEDLINE | ID: mdl-34097494

ABSTRACT

Antibiotic collateral sensitivity, in which acquired resistance to one drug leads to decreased resistance to a different drug, occurs in Burkholderia multivorans. Here, we observed that treatment of extensively drug-resistant variants evolved from a cystic fibrosis (CF) sputum sample isolate with either meropenem or sulfamethoxazole-trimethoprim, depending on past resistance phenotypes, resulted in increased sensitivity to five different classes of antibiotics. We further identified mutations, including putative resistance-nodulation-division efflux pump regulators and uncharacterized pumps, that may be involved in this phenotype in B. multivorans.


Subject(s)
Burkholderia Infections , Burkholderia cepacia complex , Burkholderia , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Burkholderia/genetics , Burkholderia Infections/drug therapy , Burkholderia cepacia complex/genetics , Drug Resistance , Humans , Microbial Sensitivity Tests
2.
Appl Environ Microbiol ; 67(11): 5325-7, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11679363

ABSTRACT

We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of Escherichia coli to enter the viable but nonculturable (VBNC) condition. Copper-induced VBNC cells could be resuscitated for up to 2 weeks after entering the VBNC state.


Subject(s)
Copper/pharmacology , Escherichia coli/drug effects , Escherichia coli/growth & development , Culture Media
3.
Appl Environ Microbiol ; 67(9): 3866-72, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11525979

ABSTRACT

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.


Subject(s)
Betaproteobacteria/growth & development , Plant Diseases/microbiology , Solanum lycopersicum/microbiology , Betaproteobacteria/pathogenicity , Copper Sulfate/metabolism , Culture Media , Soil Microbiology , Virulence
4.
Urol Res ; 29(1): 60-6, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11310218

ABSTRACT

Involvement of the viable but nonculturable (VBNC) condition in recurrent urinary tract infections (UTIs) was investigated. VBNC bacteria are those which are alive but do not give rise to visible growth under nonselective growth conditions. Urine, bladder, and kidney samples collected over a 2-month period from BALB/c mice inoculated with the uropathogenic Escherichia coli strain J96 were examined to determine the level of culturable and viable bacteria. Urine from uninoculated mice was found to contain more viable than culturable bacteria. Inoculated mice had a transient increase in the level of culturable forms of the uropathogen in their urine, followed by a decrease to background levels; they also had multiple log higher levels of viable cells than culturable cells. The culturable pathogenic bacteria in mice that were inoculated and received antibiotic treatment dropped to undetectable levels within 1 week. At 2 out of 12 subsequent time points spanning an additional 65 days, culturable forms of the inoculated pathogenic bacteria were recovered. Polymerase chain reaction (PCR) analysis confirmed that DNA from the inoculated bacteria was present in a sample that yielded no culturable bacteria. These data indicate that the inoculated uropathogenic E. coli was not eliminated by antibiotic therapy, and suggest that these bacteria may escape detection by current standard culturability assays because they are VBNC.


Subject(s)
Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Escherichia coli/isolation & purification , Urinary Tract Infections/drug therapy , Urinary Tract Infections/microbiology , Animals , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents, Urinary/pharmacology , DNA, Bacterial/analysis , Escherichia coli/genetics , Escherichia coli/growth & development , Female , Kidney/microbiology , Mice , Mice, Inbred BALB C , Microbiological Techniques , Pyelonephritis/drug therapy , Pyelonephritis/microbiology , Sulfamethoxazole/pharmacology , Trimethoprim/pharmacology , Urinary Bladder/microbiology , Urine/microbiology
5.
Appl Environ Microbiol ; 65(8): 3754-6, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10427081

ABSTRACT

Many bacteria respond to changes in environmental conditions by entering the viable-but-nonculturable state. We have determined that copper can induce nutrient-starved Agrobacterium tumefaciens and Rhizobium leguminosarum cells to become viable but nonculturable. This is the first report of a chemical inducer of this condition.


Subject(s)
Agrobacterium tumefaciens/drug effects , Agrobacterium tumefaciens/growth & development , Copper/pharmacology , Rhizobium leguminosarum/drug effects , Rhizobium leguminosarum/growth & development , Agrobacterium tumefaciens/isolation & purification , Ecosystem , Rhizobium leguminosarum/isolation & purification , Water Microbiology
7.
Proc Natl Acad Sci U S A ; 91(8): 2994-8, 1994 Apr 12.
Article in English | MEDLINE | ID: mdl-8159693

ABSTRACT

During the inception of crown gall tumorigenesis, the transferred DNA (T-DNA) is processed from the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens and is transferred to plant cells. T-DNA processing and transfer require the induction of vir (virulence) genes by phenolic compounds secreted by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and double-stranded forms of processed T-DNA accumulate in the bacteria. Although current models favor the transfer of T-strands to plants, there has yet been no experimental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. tumefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that single-stranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control experiments show that this T-DNA does not originate from lysed contaminating bacterial cells. T-DNA transfer depends on a functional bacterial virB operon. Protoplast infections using an A. tumefaciens virE mutant result in a low level of accumulation of T-strands in the plant cells.


Subject(s)
Agrobacterium tumefaciens/genetics , DNA, Single-Stranded/metabolism , Gene Transfer Techniques , Base Sequence , Cells, Cultured , DNA Primers/chemistry , Genes, Bacterial , In Vitro Techniques , Molecular Sequence Data , Plants, Toxic , Plasmids , Nicotiana
9.
Mol Microbiol ; 10(3): 473-81, 1993 Nov.
Article in English | MEDLINE | ID: mdl-7968526

ABSTRACT

The relative abundance of 88 proteins was measured in extracts from three strains of Escherichia coli K-12 that are isogenic except for the topA and gyrB genes. Mutations in these genes slightly raise or lower, respectively, steady-state DNA supercoiling levels but have little effect on growth rate. Altered protein abundances were observed in the mutant strains relative to wild type. Many proteins exhibited minimum abundance at wild-type supercoiling levels, and other proteins exhibited maximal abundance at relaxed levels. A smaller number showed maximal abundance at elevated levels of supercoiling. These data suggest that small, non-lethal changes in DNA supercoiling can have widespread effects on patterns of gene expression.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type I/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , DNA Topoisomerases, Type I/physiology , DNA Topoisomerases, Type II/physiology , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Escherichia coli/genetics , Mutagenesis
10.
Nucleic Acids Res ; 18(23): 6953-8, 1990 Dec 11.
Article in English | MEDLINE | ID: mdl-2263456

ABSTRACT

Virulence genes virD1 and virD2 are required for T-DNA processing in Agrobacterium tumefaciens. The regions within virD2 contributing to T-DNA processing and virulence were investigated. Some insertional mutations in virD2 prevented T-DNA border endonucleolytic cleavage and produced an avirulent phenotype. However, a non-polar insertion immediately after bp 684 of the 1344 bp open reading frame of virD2 did not inhibit endonucleolytic cleavage but still caused a loss of virulence. This suggested that in addition to T-DNA border cleaving activity, the VirD2 protein has another virulence function which resides in the C-terminal half of the protein. Comparative nucleotide sequence analyses of virD2 showed that the first 684 bp were 81% homologous to virD2 of an octopine Ti plasmid whereas the remaining 660 bp were only 44% homologous. A plasmid containing the virD region from octopine Ti plasmid could restore both virulence and processing to a nopaline virD2 mutant. No complementation resulted when a nopaline virD2 clone containing a region similar to eukaryotic nuclear envelope transport sequences was deleted from the 3' end. These results suggest that virD1 and only the first half of virD2 are required to encode for the T-DNA processing endonuclease, and that the 3'-half of virD2 encodes a function separate from endonuclease activity that is required for virulence.


Subject(s)
Arginine/analogs & derivatives , Bacterial Proteins/genetics , Plasmids , Rhizobium/genetics , Virulence Factors , Amino Acid Sequence , Arginine/genetics , Bacterial Proteins/metabolism , Base Sequence , Blotting, Southern , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Genes , Genetic Complementation Test , Molecular Sequence Data , Mutagenesis, Insertional , Open Reading Frames , Plants/microbiology , Sequence Homology, Nucleic Acid
11.
J Bacteriol ; 172(4): 2191-3, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2318813

ABSTRACT

Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone.


Subject(s)
Conjugation, Genetic , Genes, Bacterial , Plasmids , Rhizobium/genetics , Operon , Rhizobium/pathogenicity , Virulence/genetics
12.
Plasmid ; 23(2): 85-106, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2194232

ABSTRACT

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.


Subject(s)
Genes, Bacterial , Genes, Regulator , Operon , Plasmids , Rhizobium/genetics , Base Sequence , Cloning, Molecular/methods , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Escherichia coli/genetics , Escherichia coli/growth & development , Molecular Sequence Data , Restriction Mapping , Rhizobium/growth & development , Rhizobium/pathogenicity , Virulence
13.
Proc Natl Acad Sci U S A ; 86(7): 2133-7, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2928322

ABSTRACT

To obtain bacterial-mediated oncogenic transformation of plants, the transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is transferred to its plant host cells during infection. The initial phases of transformation involve the processing of the T-DNA in the bacterial cell after induction of the vir genes located on the Ti plasmid. The kinetics and conditions of this processing were examined and upon induction with acetosyringone up to 40% of the left and right borders of the T-DNA were cleaved. This cleavage was dependent upon virA, virG, and VirD and was rec-independent. Processed T-DNA was observed within 30 min after induction and was delayed by an increased concentration of phosphate in the induction medium. When DNA was isolated in the absence of protease treatment, the DNA fragment corresponding to the left side of the cut at both the left and right border region exhibited gel retardation, suggesting one or more "pilot" proteins may be involved in T-DNA transfer. Although the relative abundance of a processed product does not necessarily imply relative importance, the preponderance of double-stranded cleavage products suggests that double-stranded T-DNA should be considered as a possible intermediate in T-DNA transfer.


Subject(s)
Arginine/analogs & derivatives , DNA, Bacterial/genetics , Plasmids , Rhizobium/genetics , Arginine/biosynthesis , Blotting, Southern , DNA, Bacterial/isolation & purification , Genes, Bacterial , Kinetics , Mutation , Plant Tumors , Restriction Mapping
14.
J Mol Biol ; 205(2): 355-62, 1989 Jan 20.
Article in English | MEDLINE | ID: mdl-2926812

ABSTRACT

Replication of the staphylococcal plasmid pT181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein RepC. Deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting RepC protein. A shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. Single and double point mutations within these 43 base-pairs were used to determine the sequence requirement for replication within the minimal origin. Deletion mutants and point mutants were tested in replication and competition assays in vivo and in vitro, and in a RepC-mediated nicking assay.


Subject(s)
DNA Replication , DNA, Bacterial/genetics , Plasmids , Bacterial Proteins/genetics , Base Sequence , Chromosome Deletion , Molecular Sequence Data , Mutation , Staphylococcus aureus
15.
J Bacteriol ; 170(10): 4983-5, 1988 Oct.
Article in English | MEDLINE | ID: mdl-2844734

ABSTRACT

Two cases are described which indicate that RNA polymerase could alter DNA supercoiling. One occurred in a topA mutant in which abnormally high levels of plasmid supercoiling were lowered by rifampin, an inhibitor of the beta subunit of RNA polymerase. The second case involves suppression of a temperature-sensitive gyrB mutation by a rifampin-resistant allele of rpoB, the gene encoding the beta subunit of RNA polymerase. Measurements of chromosomal DNA supercoiling show that the rpoB mutation reduced DNA relaxation.


Subject(s)
DNA, Bacterial/drug effects , DNA, Superhelical/drug effects , DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , Rifampin/pharmacology , DNA Topoisomerases, Type II/physiology , Escherichia coli/drug effects , Mutation , Nucleic Acid Conformation/drug effects
17.
J Virol ; 53(1): 296-8, 1985 Jan.
Article in English | MEDLINE | ID: mdl-2981350

ABSTRACT

We have found that the burst size of bacteriophage T7 was decreased in two Escherichia coli temperature-sensitive gyrase mutants incubated at the restrictive temperature. This reduction in burst size indicates that gyrase may be required for T7 growth.


Subject(s)
DNA Topoisomerases, Type II/metabolism , Escherichia coli/growth & development , T-Phages/growth & development , Escherichia coli/enzymology , Genotype , Kinetics , Species Specificity , T-Phages/enzymology
18.
J Bacteriol ; 158(2): 397-403, 1984 May.
Article in English | MEDLINE | ID: mdl-6327603

ABSTRACT

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.


Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA, Bacterial/metabolism , DNA, Superhelical/metabolism , Escherichia coli/enzymology , Centrifugation, Density Gradient , DNA Topoisomerases, Type II/genetics , Escherichia coli/genetics , Mutation , Temperature
19.
Cell ; 36(4): 1081-8, 1984 Apr.
Article in English | MEDLINE | ID: mdl-6323018

ABSTRACT

Nucleoids isolated from a temperature-sensitive gyrB mutant of E. coli, incubated at restrictive temperatures, exhibit increased sedimentation rates and an abnormal doublet or dumbbell-shaped morphology. Shifting cells from restrictive to permissive temperature prior to nucleoid isolation leads to decreases in the percentage of doublet nucleoids and in nucleoid sedimentation rates. When nucleoids isolated from mutant cells exposed to restrictive temperature are incubated with purified gyrase, the percentage of doublet nucleoids decreases as the total number of nucleoids increases. These results, together with the demonstrated ability of gyrase to decatenate small circular DNA molecules in vitro, suggest that gyrase participates in bacterial chromosome segregation through its decatenating activity.


Subject(s)
Chromosomes, Bacterial/physiology , DNA Topoisomerases, Type II/metabolism , Escherichia coli/genetics , Mutation , DNA, Bacterial/isolation & purification , DNA, Circular/genetics , Escherichia coli/enzymology , Kinetics , Temperature
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