Biomedical subjects

S Kulakauskas

Publications and source records attributed to S Kulakauskas.

12 recordsLinked to original sources

PubMed2001

Utilization of tmRNA sequences for bacterial identification.

BACKGROUND: Ribosomal RNA molecules are widely used for phylogenetic and in situ identification of bacteria. Nevertheless, their use to distinguish microorganisms within a species is often restricted by the high degree of sequence conservation and limited probe accessibility to the target in fluorescence in situ hybridization (FISH). To overcome these limitations, we examined the use of tmRNA for in situ identification. In E. coli, this stable 363 nucleotides long RNA is encoded by the ssrA gene, which is involved in the degradation of truncated proteins. RESULTS: Conserved sequences at the 5'- and 3'-ends of tmRNA genes were used to design universal primers that could amplify the internal part of ssrA from Gram-positive bacteria having low G+C content, i.e. genera Bacillus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Listeria, Streptococcus and Staphylococcus. Sequence analysis of tmRNAs showed that this molecule can be used for phylogenetic assignment of bacteria. Compared to 16S rRNA, the tmRNA nucleotide sequences of some bacteria, for example Listeria, display considerable divergence between species. Using E. coli as an example, we have shown that bacteria can be specifically visualized by FISH with tmRNA targeted probes. CONCLUSIONS: Features of tmRNA, including its presence in phylogenetically distant bacteria, conserved regions at gene extremities and a potential to serve as target for FISH, make this molecule a possible candidate for identification of bacteria.

Bacteria↗

PubMed2000

Effects of a muramidase on a mixed bacterial community.

In bacterial communities one bacterium can influence the growth of other members of the population. These interactions may be based on nutritional factors or may occur via bacterial signaling molecules that are released in the medium. We present an example, showing that in addition to the above means of interactions, muramidases, enzymes that specifically cleave peptidoglycan chains, can also mediate interactions between bacteria. Using fluorescent in situ hybridization we demonstrate that Lactococcus lactis muramidase AcmA can hydrolyze the cell wall of Streptococcus thermophilus, without affecting viability. This intercellular activity of the lactococcal muramidase results in chain disruption of streptococci in vivo. Our data lead us to propose that chains can give growth advantages to streptococci in aerobic conditions.

Aerobiosis↗

PubMed1999

In situ detection of Escherichia coli cells containing ColE1-related plasmids by hybridization to regulatory RNA II.

A method is described for the in situ detection of individual whole fixed cells of Escherichia coli containing ColE1-related plasmids. It makes use of fluorescence in situ hybridization (FISH) and the regulatory RNA II as a target molecule for both, Cy3- and HRP-labeled olinucleotide probes. Various methods for signal amplification were compared. Probes targeting the regulatory RNA I did not result in the in situ detection of plasmid-bearing cells.

Escherichia coli↗

PubMed1998

Estimation of the state of the bacterial cell wall by fluorescent In situ hybridization.

Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.

Amidohydrolases↗

PubMed1996

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

Amino Acid Sequence↗

PubMed1995

Cloning of the ppu21IM gene using a in vivo selection method.

A genetic system enabling the in vivo selection of genes encoding the DNA-modifying enzymes was developed. A gene library is transformed into a strain harboring the restriction-modification (R-M) system which a recognition sequence is a subset of the target sequence of the DNA methyltransferase (MTase) to be cloned. If the residing MTase is temperature sensitive, the inability of transformants to grow at 42 degrees C provides a simple and convenient procedure for the isolation of new MTase-encoding genes. The feasibility of this procedure has been demonstrated by the isolation of the ppu21IM gene from a Pseudomonas putida RFL21 gene library.

Bacterial Proteins↗

PubMed1995

DNA restriction-modification systems mediate plasmid maintenance.

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.

Bacillus subtilis↗

PubMed1994

Organization and sequence of the HpaII restriction-modification system and adjacent genes.

We report the organization of the HpaII restriction and modification (R-M) system from Haemophilus parainfluenzae (recognition sequence: 5'...CCGG...3'), the sequence of the gene coding for the HpaII restriction endonuclease, and the sequence of the upstream flanking DNA. The HpaII system comprises two genes, hpaIIM, coding for the methyltransferase (MTase; 358 amino acids (aa), 40.4 kDa: product, Cm5CGG), and hpaIIR, coding for the restriction endonuclease (ENase; 358 aa, 40.9 kDa: product, C'CGG). The genes are adjacent, they have the same orientation, and they occur in the order hpaIIM then hpaIIR. The ENase bears little as sequence similarity to the isoschizomeric R.BsuFI and R.MspI ENases. Upstream of, and partly overlapping hpaIIM is the coding sequence for a 141-aa protein that resembles the very-short-patch-repair endonuclease (Vsr) of Escherichia coli. Upstream of that is the coding sequence for a protein that resembles valyl-tRNA synthetase (ValS).

Amino Acid Sequence↗

PubMed1994

Tn10-mediated inversions fuse uridine phosphorylase (udp) and rRNA genes of Escherichia coli.

Two strains carrying metE::Tn10 insertions (upstream of the udp gene) were used to isolate mutants of Escherichia coli overexpressing udp. These strains differ in their gene order; one contains an inversion between the rrnD and rrnE rRNA operons. Selection was based on the ability of overexpressed Udp to complement thymine auxotrophy. Chromosomal rearrangements that connect the udp gene and promoters of different rrn operons were obtained by this selection. Seven of 14 independent mutants selected in one of the initial strains contained similar inversions of the metE-rrnD segment of the chromosome (about 12% of its length). Another mutant contained traces of a more complicated event, inversion between rrnB and rrnG operons, which was followed by reinversion of the segment between metE and the hybrid rrnG/B operon. Similar inversions (udp-rrn) in a strain already carrying an rrnE-rrnD inversion flip the chromosomal segment between metE and rrnD/E in the opposite direction. In this case, inversions are also accompanied by duplications of the chromosomal region between the rrnA and hybrid udp-rrnD/E operons. PCR amplification with a set of oligonucleotides from the rrn, Tn5, and met genes was used for more detailed mapping. Amplified fragments of the rearranged chromosomes connecting rrnD sequences and insertion elements were sequenced, and inversion endpoints were established.

Base Sequence↗

PubMed1992

cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth.

The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.

Aerobiosis↗

PubMed1991

Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage.

An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails Tn5supF transposition to lambda-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup+. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.

Bacteriophage lambda↗

PubMed1991

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.

Alleles↗