Biomedical subjects

J E Trempy

Publications and source records attributed to J E Trempy.

At least 19 recordsLinked to original sources

PubMed2001

Expression of clpX, an ATPase subunit of the Clp protease, is heat and cold shock inducible in Lactococcus lactis.

In this study, the clpX gene and surrounding sequences were cloned and sequenced from Lactococcus lactis. The putative clpX gene encodes a 411 amino acid polypeptide with a predicted molecular weight of 45.8 kDa. Analysis of the relative levels of clpX transcript revealed that in addition to a role in proteolysis of heat damaged proteins, ClpX may also be involved in cryoprotection.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2000

Growth associated exopolysaccharide expression in Lactococcus lactis subspecies cremoris Ropy352.

A natural lactococcal isolate, Lactococcus lactis ssp. cremoris Ropy352, has been previously shown to express two phenotypically distinct exopolysaccharides (ropy and mucoid). This natural isolate was cultured on various media to explore the carbon requirements for exopolysaccharide expression. Ropy exopolysaccharide expression was optimal when grown in defined media rather than on M17-based media. Ropy352 was examined for inducible lysogenic phages. No lytic burst was observed in Ropy352 with ultraviolet light or mitomycin C for phage induction. The sugar compositions of the two phenotypically distinct exopolysaccharides were determined. The ropy exopolysaccharide is composed of galactose and glucose in the molar percents of 42 and 58%, respectively. The mucoid exopolysaccharide is composed of galactose, glucose, and mannose in the molar percents of 58, 29, and 13%, respectively. Mutational analysis revealed that mutations impairing ropy exopolysaccharide expression but not affecting mucoid exopolysaccharide expression could be isolated.

Bacteriophages↗

PubMed1999

Escherichia coli RcsA, a positive activator of colanic acid capsular polysaccharide synthesis, functions To activate its own expression.

Capsule (cps) gene expression in Escherichia coli is controlled by a complex network of regulators. Transcription of the cps operon is controlled by at least two positive regulators, RcsA and RcsB. We show here that RcsA functions to activate its own expression, as seen by the 100-fold-increased expression of a rcsA::lacZ transcriptional fusion in strains with high levels of RcsA protein, either due to a mutation in lon or due to overexpression of RcsA from a multicopy plasmid. Expression of the rcsA::lacZ fusion is increased by but not dependent on the presence of RcsB. In addition, the effects of H-NS and RcsB on the expression of rcsA are independent of each other. A sequence motif, conserved between the E. coli cps promoter and the Erwinia amylovora ams promoter and previously shown to be the RcsA-RcsB binding site, was identified in the rcsA promoter region and shown to be required for high-level expression of rcsA.

Bacterial Proteins↗

PubMed1999

A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity.

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.

ATP-Dependent Proteases↗

PubMed1998

Characterization and expression analysis of a Xanthomonas oryzae pv. oryzae recA.

Nucleotide sequence of Xanthomonas oryzae pv. oryzae (Xoo) DNA from pSM-A1 was determined and sequence analysis revealed an ORF with high homology to RecA proteins. Expression analysis using an anti-RecA antibody demonstrates that MMS treatment induces recA in Xanthomonas strains but not in an Escherichia coli harbouring cloned Xoo recA. This indicates the existence of a recA regulatory mechanism in Xanthomonas that is not function in E. coli. In Xoo, recA was highly induced by treatments with chemical mutagens, UV and peroxides, while superoxides, a thiol agent, a heavy metal and heat shock were not inducers. The increased amount of RecA induced by H2O2 or MMS treatments were due to increased transcription of recA. recA showed no growth phase or starvation regulation. The pattern of recA regulation in Xoo could play important roles in stress survival in the environment and during plant-microbe interactions.

Bacterial Proteins↗

PubMed1997

Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli.

Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon+ cells, increased 38-fold in lon+ cells carrying a Tn10::delta kan insertion mapping to 24 min on the E. coli chromosome. Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::delta kan insertion. Sequencing of both sides of the Tn10::delta kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs). A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.

ATP-Dependent Proteases↗

PubMed1997

Expression of ropy and mucoid phenotypes in Lactococcus lactis.

Strains of Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris were cultured under aerobic and anaerobic conditions on plates of whey agar, Elliker agar, and M17L agar at 15, 20, and 30 degrees C to determine the environmental conditions required for the expression of the ropy phenotype. Two strains, L. lactis ssp. cremoris Ropy 352 and L. lactis ssp. cremoris Hollandicus, exhibited two distinct polysaccharide phenotypes, ropy and mucoid. Expression of these phenotypes could be induced individually or simultaneously. The inducible nature of this response suggests that genetic regulators were present. Western blots were used to determine whether or not Lon protease and RcsA, two regulators of polysaccharide expression in Escherichia coli, were present in lactococci. Lon, a negative regulator, and RcsA, an unstable positive regulator, have been shown at the structural level to be conserved in a number of Gram-negative and Gram-positive microorganisms. The present study found evidence for structural conservation of Lon protease in lactococci. Less of the Lon-like protein was observed in the ropy strains than in the nonropy strains.

ATP-Dependent Proteases↗

PubMed1996

Identification of a second RcsA protein, a positive regulator of colanic acid capsular polysaccharide genes, in Escherichia coli.

A second form of RcsA, a positive activator of the capsular polysaccharide genes (cps), has been identified in Escherichia coli. Ferguson plot analysis suggests that the two RcsA proteins differ by size rather than by charge. Both RcsA proteins are expressed from a single rcsA gene. Detection of both RcsA proteins in delta lon cells is RcsB dependent.

Bacterial Proteins↗

PubMed1995

Polysaccharide expression in Lactococci.

Lactococcus lactis produces polysaccharide under defined environmental conditions. Three approaches are being used to identify regulators of polysaccharide synthesis in the organism. i) Two new lactococcal vectors, each of which contains a promoterless reporter gene, have been developed. They are being used to select for chromosomal insertions that affect expression of polysaccharide ii) Genetic complementation with lactococcal genomic libraries identified two classes of lactococcal genomic activities, both of which affect capsule expression in Escherichia coli iii) Lon, a highly conserved protease, is a negative regulator of polysaccharide synthesis in E. coli. Antiserum specific to Lon protease reacted with a lactococcal protein similar in size to E. coli Lon. This reaction was strongest in lactococcal strains which could not be induced to express polysaccharide.

Cloning, Molecular↗

PubMed1994

Evidence for structural conservation of Lon and RcsA.

DNA probes specific to the Escherichia coli genes encoding Lon protease and RcsA hybridized to specific DNA sequences in a number of different microorganisms. Antiserum to either E. coli protein Lon or RcsA reacted with specific proteins in these organisms. These results provide structural evidence of the presence of Lon and RcsA in organisms other than E. coli.

ATP-Dependent Proteases↗

PubMed1994

Alp suppression of Lon: dependence on the slpA gene.

We have previously found that plasmids carrying the Escherichia coli alp gene (now to be called alpA) suppress two phenotypes of a delta lon protease mutant, overproduction of capsular polysaccharide and sensitivity to UV light. Suppression of these lon phenotypes is most likely explained by the increased degradation of the Lon substrates responsible for these phenotypes. We have called this suppressing protease activity Alp protease. The Alp protease activity is detected in cells after introduction of plasmids carrying the alpA gene, which encodes an open reading frame of 70 amino acids. Insertions which abolish Alp activity interrupt this open reading frame. We have used Tn10 and lambda placMu mutagenesis to identify a chromosomal locus, slpA, that is required for alpA+ suppression of delta lon. This locus maps at 57 min, close to the chromosomal location of alpA. The expression of beta-galactosidase from a lac transcriptional fusion to slpA is increased six- to eightfold when the alpA+ gene is present on a multicopy plasmid. Therefore, AlpA acts as a transcriptional regulator of the slpA gene(s); activation of slpA transcription is necessary to suppress the phenotypes of a delta lon mutation. In an accompanying paper (J. E. Kirby, J. E. Trempy, and S. Gottesman, J. Bacteriol. 176:2068-2081, 1994), we show that neither AlpA nor SlpA is a component of the protease itself but that they are part of a regulatory cascade which leads to expression of the Alp protease.

ATP-Dependent Proteases↗

PubMed1994

Excision of a P4-like cryptic prophage leads to Alp protease expression in Escherichia coli.

The Escherichia coli K-12 alpA gene product, when overproduced from a multicopy plasmid, leads to suppression of the capsule overproduction and UV sensitivity phenotypes of cells mutant for the Lon ATP-dependent protease. This suppression has previously been shown to correlate with increased in vivo activity of a previously unknown energy-dependent proteolytic activity capable of degrading Lon substrates, the Alp protease. We show in an accompanying paper that alpA, which has homology to a short open reading frame in bacteriophage P4, acts as a positive transcriptional regulator of slpA, a gene linked to alpA and necessary for suppression of lon mutants (J. E. Trempy, J. E. Kirby, and S. Gottesman, J. Bacteriol. 176:2061-2067). The sequence of slpA suggests that it encodes an integrase gene closely related to P4 int and that both alpA and slpA are part of a cryptic P4-like prophage. AlpA expression increases SlpA synthesis. Increased SlpA leads, in turn, to the excision and loss of the cryptic prophage. Excision is dependent on integration host factor as well as on SlpA. Prophage excision is necessary but not sufficient for full expression of the Alp protease. A second function (named AHA) allows full protease expression; this function can be provided by the kanamycin resistance element from Tn903 when the element is present on a multicopy plasmid. Excision and loss of the cryptic prophage apparently allow expression of the Alp protease by inactivating a small stable RNA (10Sa RNA) encoded by the ssrA gene. The precursor of this RNA has its 3' end within the cryptic prophage; the mature 3' end lies within the prophage attL site. Inactivation of ssrA by insertional mutagenesis is sufficient to allow expression of the suppressing Alp protease, even in the presence of the cryptic prophage. Therefore, 10Sa RNA acts as a negative regulator of protease synthesis or activity, and prophage excision must inactivate this inhibitory function of the RNA.

Amino Acid Sequence↗

PubMed1993

Isolation and expression in Escherichia coli of a Xanthomonas oryzae recA-like gene.

The recA gene from the bacterium Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen, was cloned based on its ability to complement DNA repair defects of Escherichia coli recA- mutants. The Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment and, when cloned into pBR322, specifies increased methylmethanesulfonate and mitomycin C resistance to E. coli recA mutants and allows lambda red- gam- to plaque on an E. coli recA- host. An E. coli recA- strain harboring a plasmid containing the Xoo recA-like gene was shown to produce a 40-kDa protein which cross-reacted with an anti-E. coli RecA antibody. A similar molecular mass protein to RecA has been detected in several Xanthomonas pathovars using an anti-E. coli RecA antibody. Furthermore, the cloned Xoo recA was shown to hybridize to genomic DNA from various Xanthomonas pathovars, but not to genomic DNA from other bacteria species under high-stringency hybridization conditions. These results indicate the isolation of the Xoo recA gene.

Bacteriophage lambda↗

PubMed1993

Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2.

A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-type, phage-sensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phage-sensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further subcloning indicated that a single EcoRI site within the 4.5-kb region must lie within the gene or its promoter. The required 4.5-kb region was sequenced and found to code for one partial and two complete open reading frames. The gene required for complementation was functionally mapped by Tn5 mutagenesis and localized to one of the two complete open reading frames, which was designated pip (an acronym for phage infection protein). pip is 2,703 bases in length. Potential promoters start 206 and 212 bases upstream of the open reading frame. A ribosome binding site and a seven-base spacer precede the GTG (Val) translation initiation codon. The amino acid sequence deduced from the gene has 901 residues and an M(r) of 99,426. Hydropathy analysis revealed four to six potential membrane-spanning regions, one near the amino terminus and the others at the extreme carboxyl terminus. The amino terminus has characteristics of a signal sequence. The putative protein would have a 650-residue, central polar domain.

Amino Acid Sequence↗

PubMed1989

Alp, a suppressor of lon protease mutants in Escherichia coli.

Escherichia coli lon mutants lack a major ATP-dependent protease, are sensitive to UV light and methylmethane sulfonate (MMS), and overproduce capsular polysaccharide. Evidence is presented that an activity (Alp), cloned on a multicopy plasmid, can suppress the phenotypes of lon mutants. The sensitivity to UV and MMS is a reflection of the stabilization of the cell division inhibitor SulA, while the capsule overproduction arises through the stabilization of a transcriptional activator of capsule biosynthetic genes, RcsA. Multicopy alp (pAlp) suppressed capsule formation in delta lon cells, and delta lon cells containing the pAlp plasmid were resistant to MMS treatment. The MMS resistance of delta lon pAlp+ cells correlates with an increase in the degradation of SulA to that found in lon+ cells. Lon-directed degradation of SulA was energy dependent, as was the increase in degradation of SulA in delta lon pAlp+ cells. alp maps close to pheA, at 57 min on the E. coli chromosome. Although pAlp can substitute for Lon, cells lacking alp activity did not have the phenotype on a lon mutant. This study demonstrates that at least one activity, when overproduced in the cell, can substitute for Lon protease.

ATP-Dependent Proteases↗

PubMed1987

Sporulation-specific sigma factor sigma 29 of Bacillus subtilis is synthesized from a precursor protein, P31.

Evidence is presented that a sporulation-essential sigma factor of Bacillus subtilis, sigma 29, is synthesized as an inactive precursor (P31) and that its activation occurs by a developmentally regulated cleavage of 29 amino acids from the P31 amino terminus. A pulse-chase experiment demonstrated that sigma 29 was derived from a preexisting protein, with appearance of radioactively labeled sigma 29 paralleling the disappearance of labeled P31. The disappearance of pulse-labeled P31 did not occur when the experiment was done with a B. subtilis strain carrying a mutation in a locus (spoIIE) required for sigma 29, but not P31, synthesis. Microsequencing of sigma 29 protein revealed that its amino terminus originates at amino acid 30 of the P31 amino acid sequence. In order to test whether a proteolytic event alone could activate P31 to a protein with sigma 29-like properties, a fusion protein (P31*) containing most of P31 was overproduced in Escherichia coli and converted in vitro into a protein with the electrophoretic mobility of sigma 29 by limited treatment with Staphylococcus aureus V8 protease. Protease-treated P31*, but not untreated P31*, was capable of directing B. subtilis core RNA polymerase to specifically initiate RNA synthesis at a sigma 29-recognized promoter in vitro.

Amino Acid Sequence↗

PubMed1985

Bacillus subtilis sigma factor sigma 29 is the product of the sporulation-essential gene spoIIG.

Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31. This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation. spoIIG-specific RNA is present in B. subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not. A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B. subtilis strain with a mutation at the spoIIG locus (spoIIG41). The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31.

Antibodies, Monoclonal↗

PubMed1985

Synthesis of sigma 29, an RNA polymerase specificity determinant, is a developmentally regulated event in Bacillus subtilis.

Using an immunological probe, we have determined that the synthesis of the Bacillus subtilis RNA polymerase promoter specificity determinant sigma 29 is a developmentally regulated event. sigma 29 is absent from vegetatively growing cells but is abundant in sporulating cells for a restricted (2-h) period during differentiation (hour 2 to hour 4 into the sporeforming process). The narrowness of this period suggests that sigma 29 is a regulatory factor that directs the transcription of a subpopulation of genes at a precise, intermediate stage of spore formation. This view predicts that sigma 29 should be dispensable for early sporulation events. We verified this prediction by an analysis of sigma 29 accumulation in mutants that are blocked at different stages of sporulation in which we show that cells can advance to at least an intermediate point in development (stage III) in the absence of detectable sigma 29. Lastly, our anti-sigma 29 antibody probe detected a second, previously unrecognized protein in Bacillus cell extracts that may be a precursor to sigma 29. This protein, P31 (molecular weight, 31,000) is synthesized earlier in sporulation than is sigma 29. It has a peptide profile that is similar to sigma 29 and is present in all Bacillus subtilis Spo- mutants that were tested and found to still be able to accumulate sigma 29.

Antibodies, Monoclonal↗