Biomedical subjects

L J Shimkets

Publications and source records attributed to L J Shimkets.

At least 37 records · Page 2Linked to original sources

PubMed1995

Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes.

Burkholderia (Pseudomonas) cepacia is a common environmental bacterium which can be pathogenic for plants and humans. In this study, four strategies were used to identify aquatic isolates: API test strips, hybridization with species-specific DNA probes for the 16S and 23S rRNA genes, fatty acid methyl ester (FAME) profiles, and growth on selective medium (TB-T agar [C. Hagedorn, W. D. Gould, T. R. Bardinelli, and D. R. Gustarson, Appl. Environ. Microbiol. 53:2265-2268, 1987]). Only 59% of the isolates identified as B. cepacia with the API test strips were confirmed as B. cepacia by using fatty acid profiles. The 23S rRNA probe generated a few false-positive results but dramatically underestimated the number of B. cepacia isolates (i.e., 40% of the colonies that did not hybridize to the probe were B. cepacia, as determined by FAME). The 16S rRNA probe generated more false-positive results than the 23S rRNA probe but was effective in identifying the majority of the B. cepacia isolates. The selective medium was only partially successful in recovering B. cepacia. Use of the B. cepacia-specific 16S rRNA probe was the most efficient and accurate way of identifying B. cepacia.

Base Sequence↗

PubMed1995

Genetic structure of a lotic population of Burkolderia (Pseudomonas) cepacia.

The genetic structure of a population of Burkholderia (Pseudomonas) cepacia isolated from a southeastern blackwater stream was investigated by using multilocus enzyme electrophoresis to examine the allelic variation in eight structural gene loci. Overall, 213 isolates were collected at transect points along the stream continuum, from both the sediments along the bank and the water column. Multilocus enzyme electrophoresis analysis revealed 164 distinct electrophoretic types, and the mean genetic diversity of the entire population was 0.574. Genetic diversity values did not vary spatially along the stream continuum. From a canonical discriminant analysis, Mahalonobis distances (measurements of genetic similarity between populations) revealed significant differences among the subpopulations at the sediment sampling points, suggesting bacterial adaptation to a heterogeneous (or patchy) microgeographical environment. Multilocus linkage disequilibrium analysis of the isolates revealed only limited association between alleles, suggesting frequent recombination, relative to binary fission, in this population. Furthermore, the dendrogram created from the data of this study and the allele mismatch distribution are typical of a population characterized by extensive genetic mixing. We suggest that B. cepacia be added to the growing list of bacteria that are not obligatorily clonal.

Alleles↗

PubMed1994

A physical map of the Myxococcus xanthus chromosome.

A physical map of the 9.2-Mbp Myxococcus xanthus DK1622 chromosome at a resolution of 25 kbp was constructed by using a strategy that is applicable to virtually all microorganisms. Segments of the chromosome were used as hybridization probes to subdivide a yeast artificial chromosome (YAC) library into groups of linked clones. The clones were aligned by comparing their EcoRI restriction patterns. The groups of YAC clones ("contigs") were oriented and aligned with the genomic restriction map by means of common genetic and physical markers such as rare restriction sites and transposon insertions. Over 95% of the genome is represented by cloned DNA. Sixty genetic loci including > 100 genes, many of which play a role in fruiting body development, have been mapped in this way. Additional genes can now be located on the chromosome map by hybridization of their sequences to the ordered set of YAC chromosomes. The mapped genetic loci account for approximately 2% of the genome.

Base Sequence↗

PubMed1994

Cloning and characterization of the socA locus which restores development to Myxococcus xanthus C-signaling mutants.

The csgA gene produces an intercellular signal during fruiting body formation of the myxobacterium Myxococcus xanthus. Sporulating pseudorevertants were isolated to allow us to understand the mechanism by which CsgA is perceived by cells and used to regulate developmental gene expression. Two strains, LS559 and LS560, which have closely linked transposon insertions, soc-559 (formerly csp-559) and soc-560 (formerly csp-560), respectively, regained all the developmental behaviors lost by the csgA mutation including the ability to ripple, form fruiting bodies, and sporulate. The sequence analysis of the socA locus revealed that there are three putative protein-coding regions, designated socA1, socA2, and socA3. The deduced amino acid sequence of socA1 exhibits characteristics of the short-chain alcohol dehydrogenase family. The deduced amino acid sequence of socA2 shares 48% identity with the frdD gene product of the frd operon in Proteus vulgaris which anchors fumarate reductase to the membrane. The deduced amino acid sequence of socA3 does not show homology to any known proteins. Genotypic complementation, Northern (RNA) blotting, DNA sequence analysis, and the pattern of gene expression all suggest that these three genes are polycistronic. Since the socA mutations effectively bypass CsgA, the question of why csgA is maintained in M. xanthus was examined by studying the long-term stability of socA spores. Unlike the wild type, socA mutant spores germinated on starvation agar. Transmission electron micrographs of spore thin sections revealed that germination is not due to an obvious structural deficiency of the socA spores. These results suggest that the ability of socA myxospores to survive long periods under unfavorable environmental conditions is severely comprised. Therefore, soxA appears to be essential for the development of M. xanthus.

Amino Acid Sequence↗

PubMed1993

Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA.

Resistance to kanamycin and neomycin in the bacterial assemblage of a coastal plain stream was detected by growth of colonies on media containing antibiotics. Three of 184 kanamycin-resistant colonies hybridized with a probe containing the nptII gene from transposon Tn5; the nptII gene encodes the enzyme neomycin phosphotransferase and conveys resistance to kanamycin and neomycin. In one of these isolates, the homologous gene was cloned and shown to confer resistance to a kanamycin-sensitive Escherichia coli strain. Since enumeration of bacteria by acridine orange direct counts revealed that less than 0.2% of the bacteria present were cultivated, direct examination of environmental DNA was used to assess abundance of sequences that hybridize to the nptII gene. To examine the resistance potential of bacteria that were not cultured, total DNA was extracted from environmental samples and hybridized with specific probes. The relative amount of eubacterial DNA in each sample was determined by using a eubacterial specific rDNA probe. Then, the abundance of sequences that hybridize to the eubacterial neomycin phosphotransferase gene was determined by hybridization and expressed relative to the total eubacterial DNA in the assemblage. Relative gene abundance was significantly different among assemblages from different habitats (leaves, midchannel sediments, and bank sediments) but did not differ among stream sites.

Base Sequence↗

PubMed1993

Regulation of cohesion-dependent cell interactions in Myxococcus xanthus.

Myxococcus xanthus has two nearly independent genetic systems, A and S, which appear to mediate adventurous (single-cell) movement and social (group) movement, respectively. In addition to a notable reduction in group movement, social motility mutants exhibit decreased biofilm formation, cell cohesion, dye binding, fibril production, and fruiting body formation. The stk-1907 allele, containing transposon Tn5 insertion omega DK1907, was introduced into wild-type cells and many social motility mutants. This allele, which was epistatic to most social motility mutations, caused wild-type and most mutant cells to exhibit increased group movement, cell cohesion, dye binding, and production of cell surface fibrils. The presence of the stk-1907 allele in dsp mutants, which almost completely lack cell surface fibrils, did not result in these phenotypic changes; therefore, stk-1907 is hypostatic to dsp mutations. Those mutants which exhibited increased group movement and cell cohesion with the stk-1907 allele also had increased fruiting body formation, but no significant changes in spore production were observed. These results suggest that fibrils may mediate cell cohesion, dye binding, and group movement. Additionally, the results suggest that the dsp locus contains genes involved in subunit synthesis, transport, and/or assembly of fibrils. The wild-type and mutant alleles of stk were cloned and studied in merodiploids. The mutant allele is recessive, suggesting that Tn5 omega DK1907 caused a null mutation in a gene which acts as a negative regulator of fibril synthesis. The stk-1907 allele appears to cause utilization of the A motility system for group movement, possibly because of increased fibril production.

Agglutination Tests↗

PubMed1993

Effect of dsp mutations on the cell-to-cell transmission of CsgA in Myxococcus xanthus.

The dsp locus contains genes involved in the subunit synthesis and/or assembly of fibrils that radiate outward from the Myxococcus xanthus cell surface and attach to other cells. The csgA gene encodes an extracellular protein morphogen which is essential for fruiting body development. The question of whether fibrils are involved in the transmission of CsgA to adjacent cells was investigated in three ways. First, the dsp and csgA mutants were mixed in a ratio of 1:1 and allowed to develop; fruiting bodies containing spores derived from the csgA mutant were formed, suggesting efficient CsgA transfer. Second, the csgA mutation affected expression of many developmentally regulated genes differently from the way dsp affected their expression. Third, the expression of one developmentally regulated gene, which was partially expressed in csgA and dsp backgrounds, was almost completely inhibited in the presence of both mutations, suggesting that its promoter is regulated independently by two distinct stimuli, one that is csgA dependent and one that is dsp dependent. Together these results argue that fibrils are not necessary for cell-to-cell transmission or perception of CsgA, and their precise function remains unknown.

Bacterial Adhesion↗

PubMed1992

csgA expression entrains Myxococcus xanthus development.

The development cycle of the myxobacterium Myxococcus xanthus consists of three partially overlapping morphological stages referred to as rippling, fruiting body formation, and sporulation, all of which are absent in csgA null mutants. The CsgA gene product is an extracellular protein, referred to as the C signal, which is essential for developmental cell-cell interactions. csgA expression increases throughout development, reaching its peak during sporulation. CsgA was made limiting for development by constructing nested deletions upstream from the csgA gene, which resulted in reduced csgA expression. Successively larger deletions resulted in termination of development at earlier and earlier stages, with rippling requiring approximately 20% maximum csgA expression, fruiting body formation requiring approximately 30% expression, and sporulation requiring 82% expression. Conversely, artificial induction of csgA also induced development provided nutrients were limiting. These results suggest that steady increases in CsgA over the course of development entrain the natural sequence of morphological events. The csgA upstream region appears to process information concerning the levels of nutrients, peptidoglycan components, and the B signal. In the absence of nutrients, a region extending 400 bp upstream from the start site of transcription was necessary for development and maximal csgA expression. In the presence of low levels of nutrients, a region extending approximately 930 bp upstream was essential for the same tasks. It appears that the upstream region extending from -400 to -930 stimulates csgA expression in the presence of excess carbon, nitrogen, and phosphate, thereby allowing development to go to completion.

Base Sequence↗

PubMed1991

Physical map of the Myxococcus xanthus chromosome.

The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.

Blotting, Southern↗

PubMed1991

Low-temperature induction of Myxococcus xanthus developmental gene expression in wild-type and csgA suppressor cells.

The csgA gene encodes an extracellular protein that plays an essential role in the regulation of fruiting-body formation and sporulation of Myxococcus xanthus. The csgA suppressor allele soc-500 (formerly referred to as csp-500) was selected based on its ability to restore sporulation to csgA cells under developmental conditions at 32 degrees C. The soc-500 allele was subsequently found to induce sporulation of csgA+ or csgA cells simply by shifting the temperature of vegetatively growing cells to 15 degrees C. Low-temperature-induced sporulation of soc-500 strains occurred in the absence of two requirements for fruiting-body sporulation: low nutrient levels and a high temperature. Low temperature alone caused the expression of many developmentally regulated genes but did not support the development of wild-type cells. The soc-500 allele appears to activate genes involved with sensing nutritional stress. At low temperature on a nutritionally rich medium, soc-500 induced expression of the tps gene which is normally expressed following nutritional shiftdown. The soc-500 allele was cloned and integrated into the wild-type chromosome by site-specific recombination. It was dominant over the wild-type allele in merodiploids and is contained on a 3-kbp DraI-ClaI restriction fragment. The soc-500 transcriptional unit spans a 300-bp PstI-PstI restriction fragment, since deletion of the PstI restriction fragment inhibits both csgA suppression and low-temperature induction. These results suggest that the soc-500 mutation lies in a gene that is involved in nutrient sensing.

Alleles↗

PubMed1990

Nucleotide sequence and transcriptional products of the csg locus of Myxococcus xanthus.

The csg locus of Myxococcus xanthus appears to control the production of an intercellular signal that is essential for development. The complete nucleotide sequence of a clone containing the csg locus was determined by the dideoxy-chain termination method. Pattern recognition analyses of the DNA sequence revealed the presence of two protein-coding regions that are convergently oriented and separated by only 8 nucleotides. Tn5 lac insertions into this clone detected two transcriptional units that are transcribed in a convergent fashion and whose expression increases during development. The two genes represented by these protein-coding regions and transcriptional units have been designated csgA and fprA. Northern (RNA) blot analyses detected an 800-nucleotide RNA specific to the csgA gene and a 900-nucleotide RNA specific to the fprA gene. Our results, along with mutational studies, identify csgA as the gene involved in cell communication. The function of the fprA gene is described in an accompanying paper (L. J. Shimkets, J. Bacteriol. 172:24-30, 1990).

Bacterial Proteins↗

PubMed1990

The Myxococcus xanthus FprA protein causes increased flavin biosynthesis in Escherichia coli.

The fprA gene is immediately adjacent to the csgA gene (formerly known as spoC) of Myxococcus xanthus. Whereas the csgA gene has an essential role in cell interactions during the developmental cycle, the function of the fprA gene is unknown. Gene disruption was used to determine what affect a null mutation in this gene has on the phenotype of the cell. A csgA-fprA deletion and an fprA frameshift mutation were constructed in vitro in a cloned copy of this locus and then inserted into the M. xanthus chromosome to create a merodiploid with the wild-type and mutant alleles in tandem. The merodiploid was then allowed to segregate one of the two alleles along with the vector sequences in an effort to replace the wild-type allele with the mutant allele. All of the segregants had the wild-type allele, suggesting that a functional fprA gene is essential for vegetative growth. The fprA gene was placed under control of the lacZ transcriptional and translational signals and overexpressed in Escherichia coli, and the new host was examined for any phenotypic changes. A 27-kilodalton protein was observed in sodium dodecyl sulfate-polyacrylamide gels of total-cell protein as predicted from the DNA sequence of this gene. Overexpression of FprA caused the accumulation of a yellow pigment with spectral and redox properties similar to that of the flavins. The pigment cochromatographed with flavin mononucleotide by Silica Gel G thin-layer chromatography. Approximately two-thirds of the total cellular flavin was associated with soluble protein. The major soluble flavin-associated protein was purified on DEAE-Bio-Gel A and Phenyl-Sepharose CL-4B and by polyacrylamide gel electrophoresis. The amino acid composition of the purified protein was similar to that predicted from the DNA sequence of the FprA fusion protein. Apparently, overproduction of FprA (for flavin-associated protein A) in E. coli resulted in a large increase in flavin biosynthesis. Together, these results suggest that the fprA gene encodes a protein that is associated with flavin mononucleotide and has an essential function in M. xanthus.

Amino Acids↗

PubMed1990

Genome size of Myxococcus xanthus determined by pulsed-field gel electrophoresis.

Genomic DNA of the myxobacterium Myxococcus xanthus was digested with the rare cutting restriction endonuclease AseI or SpeI, and the restriction products were separated by pulsed-field gel electrophoresis. Transposons Tn5-132 and Tn5 lac, which contain AseI restriction sites, were used to determine the number of restriction fragments in each band. The size of the genome was determined by adding the molecular sizes of the restriction products. The genomes of strains DK101, MD2, and DZF1 have identical restriction patterns and were estimated to be 9,454 +/- 101 kilobase pairs from the AseI digestions and 9,453 +/- 106 kilobase pairs from the SpeI digestions. DK1622, which was derived from DK101 by treatment with UV light, has suffered a 220- to 222-kilobase-pair deletion that removed an AseI and an SpeI restriction site. The deleted DNA may consist exclusively of Mx alpha-associated sequences.

DNA Transposable Elements↗

PubMed1990

CsgA, an extracellular protein essential for Myxococcus xanthus development.

CsgA mutants of Myxococcus xanthus appear to be defective in producing an extracellular molecule essential for the developmental behaviors of this bacterium. The csgA gene encodes a 17.7-kilodalton polypeptide whose function and cellular location were investigated with immunological probes. Large quantities of the CsgA gene product were obtained from a lacZ-csgA translational gene fusion expressed in Escherichia coli. The chimeric 21-kilodalton protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity-purified polyclonal antibodies raised against the fusion protein were used to determine the cellular location of the native CsgA protein by colloidal gold labeling and transmission electron microscopy. Between 1,100 and 2,200 extracellular molecules of CsgA per developing M. xanthus cell were detected, most of which were associated with the extracellular matrix. The anti-CsgA antibodies inhibited wild-type development unless they were first neutralized with the fusion protein. Together these results suggest that the CsgA gene product has an essential, extracellular function during development, possibly as a pheromone.

Bacterial Proteins↗

PubMed1990

Social and developmental biology of the myxobacteria.

Myxobacteria are soil bacteria whose unusually social behavior distinguishes them from other groups of procaryotes. Perhaps the most remarkable aspect of their social behavior occurs during development, when tens of thousands of cells aggregate and form a colorful fruiting body. Inside the fruiting body the vegetative cells convert into dormant, resistant myxospores. However, myxobacterial social behavior is not restricted to the developmental cycle, and three other social behaviors have been described. Vegetative cells have a multigene social motility system in which cell-cell contact is essential for gliding in multicellular swarms. Cell growth on protein is cooperative in that the growth rate increases with the cell density. Rippling is a periodic behavior in which the cells align themselves in ridges and move in waves. These social behaviors indicate that myxobacterial colonies are not merely collections of individual cells but are societies in which cell behavior is synchronized by cell-cell interactions. The molecular basis of these social behaviors is becoming clear through the use of a combination of behavioral, biochemical, and genetic experimental approaches.

Base Sequence↗

PubMed1989

The role of the cell surface in social and adventurous behaviour of myxobacteria.

The myxobacteria are an unusually social group of prokaryotic organisms that form fruiting bodies containing dormant myxospores in response to nutritional stress. Social behaviour is controlled by a multigene system known as 'S' and by a series of intercellular signals that are released during development. The genes controlling these communication systems have been identified by mutational analysis and current research is directed toward examining the functions of these genes. S- mutants are generally nondevelopmental and noncohesive. They lack pili, a Congo red receptor, and 50-nm-wide fibrils which extend outward from the cell surface. Changes in the architecture of the cell surface have been studied by means of surface labelling and with monoclonal antibodies directed against cell-surface antigens. The cell surface undergoes dramatic changes during the course of development. Most vegetative antigens decrease in concentration or disappear completely while new development-specific antigens appear.

Antigens, Bacterial↗

PubMed1989

Developmental bypass suppression of Myxococcus xanthus csgA mutations.

The csgA mutations of Myxococcus xanthus (formerly known as spoC) inhibit sporulation as well as rippling, which involves ridges of cells moving in waves. Sporulating revertants of CsgA cells were isolated by direct selection, since spores are much more resistant to heat and ultrasonic treatment than are vegetative cells. The revertants fell into seven groups on the basis of phenotype and the chromosomal location of the suppressor alleles. Group 1 contained one allele that was a back mutation of the original csgA mutation. Group 2 contained two linked alleles that were unlinked to the csgA locus and restored fruiting-body formation, sporulation, and rippling. Group 3 revertants regained the ability to sporulate in fruiting bodies but not the ability to ripple. Revertants in groups 4 to 7 were able to sporulate but unable to form fruiting bodies or ripples. The suppressors were all found to be bypass suppressors even though they were not selected as such in most cases. The csgA mutation prevented expression of several developmentally regulated promoters, each fused to a lacZ reporter gene and assayed by beta-galactosidase production. In four of five suppressor groups (groups 4 to 7), expression of each of these csgA-dependent fusions was restored, which suggests that bypass suppression restores developmental gene expression near the point at which expression is disrupted in CsgA mutants. Bypass suppression did not restore production of C factor, and morphological manifestations of development such as rippling and fruiting-body formation were usually abnormal. One interpretation of these results is that C factor has multiple functions and few suppressors can compensate for all of them.

Cell Differentiation↗

PubMed1988

Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus.

The myxobacteria are among the simplest organisms with a developmental cycle that is dependent on cell cooperation, and they provide an outstanding system with which to study genes involved in cell interactions. Myxococcus xanthus cells which acquire a csg mutation (formerly known as spoC) lose three different traits, the ability to sporulate, the ability to stimulate adjacent Csg cells to sporulate, and the ability to ripple. The boundaries of the csg locus were determined by transferring a recombinant DNA molecule containing all or part of the locus to Csg mutants and examining the sporulation and rippling phenotypes of the transductants. Three methods were used to integrate the csg locus into the chromosome. First, the entire molecule was integrated into the chromosome by a single homologous crossover. Second, a portion of the molecule was integrated into the chromosome by two flanking homologous crossovers. Third, the entire molecule was integrated into the chromosome by site-specific recombination at a bacteriophage attachment site. Together, these techniques suggested that all of the functions of the csg locus are carried on a DNA fragment of 1.9 kbp or less. The locus appears to contain two smaller units of function. Transposon insertions or deletions in the right end of the locus disrupted sporulation and intercellular complementation of Csg mutants for sporulation, but did not disrupt rippling. The intercellular complementation of Csg mutants may reflect a natural and necessary step in the sporulation of wild-type cells, since the ability to sporulate and the ability to stimulate Csg mutants to sporulate were inseparable by any of these methods.

DNA Transposable Elements↗