Stress response in Lactococcus lactis.
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Base Sequence↗
Biomedical subjects
C A Batt
Publications and source records attributed to C A Batt.
Cloning and sequencing of the Lactococcus lactis subsp. lactis dnaK gene using a PCR-based approach.
The coding region for the dnaK gene from Lactococcus lactis subsp. lactis LM0230 was isolated and sequenced. An internal 789-bp fragment was amplified by the polymerase chain reaction (PCR) using a pair of degenerate oligodeoxyribonucleotide primers designed on the basis of amino acid (aa) sequences conserved in a number of DnaK. This PCR product was cloned, sequenced and used as a Southern hybridization probe to locate the flanking regions of the gene. The sequence of this central region from dnaK was also used to design two sets of inverse PCR primers to amplify, separately, the upstream and downstream regions. The inverse PCR products were then cloned and partially sequenced. The complete nucleotide sequence was obtained from overlapping cloned fragments of the gene and found to consist of a single 1824-bp open reading frame coding for a 602-aa protein. Alignment of the deduced aa sequence with those of other bacterial DnaK showed a high degree of homology and is most similar to the Bacillus megaterium DnaK.
Amino Acid Sequence↗
Probing the retinol-binding site of bovine beta-lactoglobulin.
The retinol-binding site of beta-lactoglobulin has been located by selective modification of amino acid residues which reside in the two putative binding sites. Based upon two separate crystallographic analyses of bovine beta-lactoglobulin, different binding sites for retinol have been proposed: one proposal favors an interior cavity, the other a surface cleft. To discriminate between these two models, we have made four separate site-directed mutations introducing a W19A or a K70M in the interior pocket and a F136A or K141M in the surface pocket. The K70M beta-lactoglobulin exhibited a marked decrease in its binding of retinoic acid compared to the F136A, K141M, and wild-type proteins. Retinyldenepropylamine, a retinyl Schiff base analog of retinol, was synthesized and its absorption spectrum when bound to the wild-type, K70M, and K141M proteins was examined to probe its interaction with the respective lysine residues. The retinylidenepropylamine bound in the K70M beta-lactoglobulin exhibited a kinetic red shift as distinct from the blue shift observed when it is bound to either the K141M or wild-type beta-lactoglobulins. The blue shift indicates protonation of the Schiff base. The resulting tagged peptide was isolated after cyanogen bromide cleavage and found to be the Ala25-Met107 peptide, consistent with the Lys70 being the residue which interacts with the bound retinol. These results support the proposal that retinol binds to an evolutionarily conserved interior cavity rather than the surface pocket.
Amino Acid Sequence↗
Perturbing the metal site in D-xylose isomerase. Effect of mutations of His-220 on enzyme stability.
The histidine residue at position 220 in the Streptomyces rubiginosus D-xylose isomerase is conserved in all D-xylose isomerases. The three-dimensional structure of D-xylose isomerase reveals that His-220 is part of the octahedral coordination sphere of M2, one of two metal ions (Mn2+) in the active site. This work describes the effects of replacing His-220 with Ser, Glu, Asn, and Lys. The consequences of these amino acid substitutions on enzyme activity, thermostability, and structure were analyzed by kinetic, denaturation, and crystallographic methods. The kcat values H220S, H220N, and H220E are only 0.3-0.5% of the wild-type values, and the Km for each of these mutant enzymes increased by 30-40-fold over the wild-type value. The mutant enzyme H220K did not exhibit any measurable activity. Thermal denaturation studies (Tm values) indicate that the H220S and H220N mutant enzymes are approximately 5-8 degrees C less stable than the wild-type enzyme, whereas H220E and H220K are 13-24 degrees C less stable than the wild-type enzyme. To analyze the molecular basis for this decreased thermostability, the crystal structures of the H220S, H220N, and H220E mutant enzymes complexed with Mn2+ have been determined at 1.95, 1.90, and 1.75 A, respectively. In the H220S structure, a water molecule effectively replaces the N epsilon-2 atom of the imidazole ring of His-220 and mediates the interaction between Mn2+ at the M2 site and Ser-220. A similar water-mediated interaction between the metal ion and Asn-220 is observed in H220N. No direct or water-mediated interactions between the carboxyl group of Glu-220 and the metal are observed in H220E. Whereas octahedral coordination is maintained for the metal at the M2 site in H220S and H220N, a pentahedral coordination with the metal at the M2 site is observed in H220E. Metal activation measurements support the observation that metal binding is perturbed and is responsible for thermal lability of His-220 mutants.
Aldose-Ketose Isomerases↗
Thermostable variants of bovine beta-lactoglobulin.
The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond. Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121. We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C. Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein. These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method. Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping. In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized. The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.
Amino Acid Sequence↗
Ligase chain reaction (LCR)--overview and applications.
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Animals↗
Identification of Erwinia stewartii by a ligase chain reaction assay.
A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed such that only E. stewartii DNA gave a product in the LCR assay. The ligated product was separated from the radioactively labelled primers by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Twenty-four different Erwinia species and strains were tested by PCR-coupled LCR to verify the specificity of the assay, and only E. stewartii strains gave a positive reaction. In addition, infected and healthy plant material was also assayed. E. stewartii was detected in infected plant material, even when large populations of epiphytic bacteria were present. No enrichment was necessary for detection of the pathogen in corn leaves. This assay has potential as a diagnostic technique for the detection of E. stewartii in infected plant and vector material.
Base Sequence↗
Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants.
Two outbreaks of epizootic listerial encephalitis, one in sheep and one in goats, were investigated through pathology, microbiology, and DNA amplification-based techniques. Efforts were made to survey the diversity of Listeria monocytogenes strains in the silage consumed by affected animals and to verify the causal relationship between silage and disease outbreak. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue samples. Random amplified polymorphic DNA patterns revealed two distinct L. monocytogenes strains, one of which was identical to the sheep brain isolate, in the silage associated with the outbreak in sheep. Three brain isolates and one silage isolate, all of which had different random amplified polymorphic DNA patterns, were found in the outbreak involving goats. All isolates from both outbreaks were indistinguishable in an in vitro assay for cell-to-cell spread and growth in macrophages. All brain isolates from the goat outbreak had identical intracellular ActA patterns, which were different from the pattern for the silage isolate. While the sheep brain isolate had an ActA pattern different from that of the corresponding silage isolate, the patterns for the brain isolates from the two outbreaks were not identical. This survey demonstrates the diversity of L. monocytogenes in silage and suggests the existence of one or more selective processes by which certain strains are more prone to give rise to disease.
Animals↗
Verification of causal relationships between Listeria monocytogenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified polymorphic DNA patterns.
Food and clinical isolates of Listeria monocytogenes recovered from four different outbreaks of listeriosis were analyzed by their PCR-based randomly amplified polymorphic DNA (RAPD) patterns to verify their causal relationships. The generation of DNA fingerprints by PCR-based RAPD analysis is a fast and sensitive method for the epidemiological tracking and identification of bacteria implicated in food poisoning outbreaks. The L. monocytogenes strains used in the study were obtained from the following four outbreaks: California, 1985, Mexican-style cheese; Canadian Maritime Provinces, 1981, coleslaw; Canada, 1989, brie cheese; and Canada, 1989, alfalfa tablets. RAPD profiles were generated by using random 10-mer primers for at least one food and one clinical isolate recovered from each outbreak. Identical profiles for 20 different primers were observed for each pair of food and clinical isolates from two of the four outbreaks. Isolates from the outbreak involving alfalfa tablets exhibited identical patterns for 19 primers; however, primer OPA-1 produced one additional 1.8-kb fragment, designated OPA-1-1.8, that was found in the food isolate but not in the corresponding clinical isolate. Hybridization analysis revealed that the absence of the OPA-1-1.8 polymorphic fragment in the clinical isolate was due to a deletion of at least 1.8 kb. Loss of the OPA-1-1.8 polymorphic fragment could not be induced by infective passage of the L. monocytogenes isolate from the alfalfa tablet through a mouse or by growth of this isolate under selective conditions. This suggests that the isolate recovered from the food was not identical to the isolate recovered from the patient. The ability to produce unique RAPD patterns allows for the discrimination between isolates even if they are of the same serotype and multilocus enzyme electrophoretic type.
Animals↗
Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction.
A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3' end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5' biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3'-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos 530 and the entire assay including DNA isolation can be completed within 8 h.
Animals↗
Cloning and sequencing of the Lactococcus lactis subsp. lactis groESL operon.
The operon (groESL) coding for the Lactococcus lactis subsp. lactis heat-shock proteins GroEL and GroES, has been isolated and its complete nucleotide (nt) sequence determined. A set of degenerate PCR primers, deduced from amino acids which are conserved in a number of prokaryotic GroELs, were synthesized and used to amplify a 957-bp fragment. This PCR fragment was used as a probe to isolate a 5.0-kb EcoRI chromosomally derived fragment. A region of this 5.0-kb EcoRI fragment was sequenced and revealed that the groES gene was located 5' to groEL. This sequence was then used to design a set of inverse PCR primers and a 2.5-kb HindIII fragment was cloned which contained the region 5' to groEL. The complete nt sequence of the groESL operon was determined from overlapping fragments. It revealed that the groESL operon was preceded by a stem-loop structure and the promoter appears similar to most L. lactis subsp. lactis and other Gram+ bacterial promoters. Northern analysis demonstrated that the groESL operon is under tight regulation and a dramatic induction of mRNA synthesis occurs within 15 min after heat shock.
Amino Acid Sequence↗
Non-radioactive detection of Mycobacterium tuberculosis LCR products in a microtitre plate format.
As part of the development of the ligase chain reaction (LCR) into a tool which can be used by a wide variety of researchers, we have investigated several analytical detection systems for the products of this amplification reaction. While early work with this technology has used gel electrophoresis to separate the LCR probes from the ligated product, solid phase capture techniques are also applicable, particularly when one of the probes is modified with a 'hook' such as biotin, and the adjoining probe modified with a detectable label. In this study we report a comparison of eight different non-radioactive detection techniques and discuss the analytical sensitivity of each. Detection with laser scanning fluorescent gel electrophoresis remains the most sensitive, with the assay described herein capable of detecting 100 molecules of the Mycobacterium tuberculosis insertion element IS6110 in a background of 4 micrograms of unrelated DNA. This method was followed closely by solid-phase capture and chemiluminescence detection which gave a sensitivity of 1000 molecules of IS6110. Fluorescence detection was approximately 10-fold less sensitive than chemiluminescence detection, and absorbance detection was a further 10-fold less sensitive than fluorescence detection. However, absorbance detection even at this level can still be useful for systems where visual interpretation is desired.
Alkaline Phosphatase↗
A combined modified reverse dot-blot and nested PCR assay for the specific non-radioactive detection of Listeria monocytogenes.
A modified reverse dot-blot assay was developed and used in combination with a nested PCR amplification of hly A for the specific detection of Listeria monocytogenes. The deoxynucleotides and digoxygenin-11-dUTP concentrations in the PCR were optimized for maximal sensitivity and economy. For the dot-blot hybridization, digoxygenin-labelled PCR products were directly spotted on nylon membrane-bound poly-dT tailed capture probes and the signal detection was either colorimetric or chemiluminescent. With crude cell lysates, the total assay could be completed in about 6 h with a detection limit between 2 and 25 colony forming units (cfu) per PCR. The assay was then tested for its applicability to environmental sampling of artificially contaminated aluminum surfaces. For environmental sampling, the assay requires an additional overnight enrichment step and has a sensitivity corresponding to 5 cfu per 25 cm2 of inoculated surface.
Base Sequence↗
Selection of oligodeoxynucleotide-directed mutants.
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Base Sequence↗
Detection of bovine herpesvirus-1 in bovine semen by a nested PCR assay.
A nested PCR assay targeting a portion of the glycoprotein IV gene has been developed for the detection of Bovine Herpesvirus-1 (BHV-1). Rapid and sensitive detection of the PCR products was achieved using a nonisotopic reverse dot-blot format with a visible color readout. Cross-reactivity of this PCR assay was not observed with the closely related BHV-3. The sensitivity of this assay when tested on a supernatant from a BHV-1 cell culture was approximately 4.5 TCID50 (50% tissue culture infectious dose). A procedure using the chelating resin Chelex 100 was used to prepare viral DNA from artificially inoculated samples of extended and raw semen for use in the PCR assay. In combination with nested PCR and reverse dot blot, this method allowed the detection of 5 x 10(3) TCID per 0.5 ml of semen, which is comparable to the detection in the Cornell Semen Test. The whole procedure can be completed in approximately 8 h. This assay has therefore the potential of replacing the currently available yet time consuming and costly detection methods for BHV-1 in bovine semen.
Animals↗
Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.
Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.
Base Sequence↗
Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria.
We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.
Base Sequence↗
Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay.
A polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay for the specific detection of Listeria monocytogenes (M. Wiedmann, J. Czajka, F. Barany, and C. A. Batt, Appl. Environ. Microbiol. 58:3443-3447, 1992) has been modified for detection of the LCR products with a nonisotopic readout. When a chemiluminescent or a colorimetric substrate for the nonisotopic detection of the LCR products was used, the PCR-coupled LCR gave a sensitivity of 10 CFU of L. monocytogenes. The detection method with the chemiluminescent substrate Lumi-Phos 530 permitted detection of the LCR products in less than 3 h, so that the whole assay can be completed within 10 h.
Base Sequence↗