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Functional Dissection of Escherichia coli Trigger Factor: Unraveling the Function of Individual Domains. G. Kramer, 2004.In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins . TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function . We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro . All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of  tig
dnaK
in cells lacking TF and DnaK, prevented protein aggregation in these
cells, and cross-linked to nascent polypeptides in vitro . However,
tigdnaK
cells expressing the N domain alone grew more slowly and showed less
viability than
tigdnaK
cells synthesizing either NP, NC, or full-length TF, indicating
beneficial contributions of the P and C domains to TF's chaperone
activity . In an in vitro system with purified components, none of the
TF fragments assisted the refolding of denatured D-glyceraldehyde-3-phosphate
dehydrogenase in a manner comparable to that of wild-type TF,
suggesting that the observed chaperone activity of TF fragments in
vivo is dependent on their localization at the ribosome . These
results indicate that the N domain, in addition to its function to
promote binding to the ribosome, has a chaperone activity per se and
is sufficient to substitute for TF in vivo .
Activity of Tigecycline (GAR-936) against Acinetobacter baumannii Strains, Including Those Resistant to Imipenem. María Eugenia Pachón-Ibáñez, 2004.We determined the in vitro activities of tigecycline and imipenem against 49 isolates of Acinetobacter baumannii, including those resistant to imipenem . The MIC at which 50% of the isolates were inhibited (MIC50) and the MIC90 for tigecycline and imipenem were 2 and 2 mg/liter and 32 and 128 mg/liter, respectively, with 92 and 20%, respectively, of the strains being susceptible . Tigecycline did not show bactericidal activity in the time-kill studies (n = 9 strains) . Imipenem showed bactericidal activity against seven out of nine strains . These in vitro results show that tigecycline has good in vitro bacteriostatic activity against A . baumannii, including strains resistant to imipenem . Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an  -Amylase and a Cyclodextrin-Hydrolyzing Enzyme.Sung-Jae Yang, 2004.Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13 . This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli . The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C . The substrate specificity of PFTA suggests that it possesses characteristics of both -amylase and cyclodextrin-hydrolyzing enzyme . Like typical
-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose . However, it could also attack and degrade pullulan and ß-cyclodextrin, which are resistant to
-amylase, to primarily produce panose and maltoheptaose, respectively . Furthermore, acarbose, a potent
-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes . These results confirm that PFTA possesses novel catalytic properties characteristic of both
-amylase and cyclodextrin-hydrolyzing enzyme .
Biphenyl and Benzoate Metabolism in a Genomic Context: Outlining Genome-Wide Metabolic Networks in Burkholderia xenovorans LB400. V. J. Denef, 2004.We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of Burkholderia xenovorans LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far . We conducted differential gene expression profiling during exponential growth on succinate, benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl . Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and benzoate degradation . All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells . For benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were up-regulated in biphenyl-grown cells, while the pathway for benzoate degradation via hydroxylation was up-regulated in benzoate-grown cells . The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C1 metabolic pathway genes was observed . The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest . The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays . ClpXP Protease Regulates the Signal Peptide Cleavage of Secretory Preproteins in Bacillus subtilis with a Mechanism Distinct from That of the Ecs ABC Transporter. Tiina Pummi, 2002.Identification and characterization of a suppressor mutation, sup-15, which partially restored secretion in the protein secretion-deficient Bacillus subtilis ecsA26 mutant, led us to discover a novel function of Clp protease . Inactivation of ClpP improved the processing of the precursor of AmyQ  -amylase exposed on the outer surface of the cytoplasmic membrane . A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex . In the absence of ClpXP, the transcription of the sipS, sipT, sipV, and lsp signal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation of ecs mutations . Specific overproduction of SipT enhanced the secretion . These findings extend the regulatory roles of ClpXP to protein secretion . ClpXP also influenced the processing of the lipoprotein PrsA . A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested . In contrast, Ecs did not affect transcription of the sip genes, pointing to a different mechanism of secretion regulation .
The Virulence Activator AphA Links Quorum Sensing to Pathogenesis and Physiology in Vibrio cholerae by Repressing the Expression of a Penicillin Amidase Gene on the Small Chromosome. Gabriela Kovacikova, 2003.Activation of the tcpPH promoter on the Vibrio pathogenicity island by AphA and AphB initiates the Vibrio cholerae virulence cascade and is regulated by quorum sensing through the repressive action of HapR on aphA expression . To further understand how the chromosomally encoded AphA protein activates tcpPH expression, site-directed mutagenesis was used to identify the base pairs critical for AphA binding and transcriptional activation . This analysis revealed a region of partial dyad symmetry, TATGCA-N6-TNCNNA, that is important for both of these activities . Searching the V . cholerae genome for this binding site permitted the identification of a second one upstream of a penicillin V amidase (PVA) gene on the small chromosome . AphA binds to and footprints this site, which overlaps the pva transcriptional start, consistent with its role as a repressor at this promoter . Since aphA expression is under quorum-sensing control, the response regulators LuxO and HapR also influence pva expression . Thus, pva is repressed at low cell density when AphA levels are high, and it is derepressed at high cell density when AphA levels are reduced . Penicillin amidases are thought to function as scavengers for phenylacetylated compounds in the nonparasitic environment . That AphA oppositely regulates the expression of pva from that of virulence, together with the observation that PVA does not play a role in virulence, suggests that these activities are coordinated to serve V . cholerae in different biological niches . Gene Dosage Effect of L-Proline Biosynthetic Enzymes on L-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae. Yukiyasu Terao, 2003.We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae . A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding  -glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn) . Interestingly, this mutation enhanced the activities of
-glutamyl kinase and
-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo . Here, we found that the Asp154Asn mutant
-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex . We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including
 1-pyrroline-5-carboxylate reductase, which converts
1-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain . Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature . However, co-overexpression of the mutant
-glutamyl kinase and the wild-type
-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex . These results indicate that both enzymes, not
1-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells . A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells . Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress . The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses .
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