Biomedical subjects

Salvador Flores

Publications and source records attributed to Salvador Flores.

6 recordsLinked to original sources

PubMed2005

Adaptation for fast growth on glucose by differential expression of central carbon metabolism and gal regulon genes in an Escherichia coli strain lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Phosphoenolpyruvate (PEP) is a key intermediate of cellular metabolism and a precursor of commercially relevant products. In Escherichia coli 50% of the glucose-derived PEP is consumed by the PEP:carbohydrate phosphotransferase system (PTS) for glucose transport. PTS, encoded by the ptsHIcrr operon, was deleted from JM101 to generate strain PB11 (PTS-Glc-). PB12, a mutant derived from PB11, grows faster than the parental strain on glucose (PTS-Glc+ phenotype). This strain can redirect some of the PEP not utilized by PTS into the high yield synthesis of aromatic compounds from glucose. Here, we report a comparative transcription analysis among these strains of more than 100 genes involved in central carbon metabolism during growth on glucose. It was found that in the PTS- strains that have reduced glucose transport capacities, several genes encoding proteins with functions related to carbon transport and metabolism were upregulated. Therefore, it could be inferred that these strains synthesize autoinducers of these genes when sensing very low internal glucose concentrations, probably for scavenging purposes. This condition that is permanently present in the PTS- strains even when growing in high glucose concentrations allowed the simultaneous utilization of glucose and acetate as carbon sources. It was found that the gal operon is upregulated in these strains, as well as the aceBAK, poxB and acs genes among others. In PB12, glk, pgi, the TCA cycle and certain respiratory genes are also upregulated. A mutation in arcB in PB12 is apparently responsible for the upregulation of the TCA cycle and certain respiratory genes.

Adaptation, Biological↗

PubMed2005

Nutrient-scavenging stress response in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system, as explored by gene expression profile analysis.

The physiological role of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been studied in Escherichia coli. It has been shown that it directly or indirectly regulates the activity of most catabolic genes involved in carbohydrate transport. Accordingly, strains lacking PTS have pleiotropic phenotypes and are impaired in their capacity to grow on glucose and other PTS sugars. We have previously reported the characterization of a mutant harboring a pts operon deletion (PB11) which, as expected, showed a severe reduction of its growth capacity when incubated on glucose as carbon source, as compared to that of the isogenic wild-type strain. These observations corroborate that PTS is the main determinant of the capacity to grow on glucose and confirm the existence of other systems that allow glucose utilization although at a reduced level. To explore the physiological state and the metabolic pathways involved in glucose utilization in a pts(-) background, we analyzed the global transcriptional response of the PB11 mutant when growing in minimal medium with glucose as carbon source. Genome-wide transcriptional analysis using microarrays revealed that, under this condition, expression of several genes related to carbon transport and metabolism was upregulated, as well as that of genes encoding transporters for certain nucleotides, nitrogen, phosphorus and sulfur sources. In addition, upregulation of rpoS and several genes transcribed by this sigma subunit was detected. These results indicate that the reduced capacity of glucose utilization present in the PB11 strain induces a general nutrient-scavenging response and this behavior is not dependent on a functional PTS. This condition is responsible of the utilization of secondary carbon sources in the presence of glucose.

Biological Transport↗

PubMed2004

Growth-rate recovery of Escherichia coli cultures carrying a multicopy plasmid, by engineering of the pentose-phosphate pathway.

Expression of plasmid-encoded genes in bacteria is the most common strategy for the production of specific proteins in biotechnological processes. However, the synthesis of plasmid-encoded proteins and plasmid-DNA replication often places a metabolic load (metabolic burden) into the cell's biochemical capacities that usually reduces the growth rate of the producing culture (Glick BR. Biotechnol Adv 1995;13:247-261). This metabolic burden may be related to a limited capacity of the cell to supply the extra demand of building blocks and energy required to replicate plasmid DNA and express foreign multicopy genes. Some of these required blocks are intermediaries of the pentose phosphate (PP) pathway, e.g., ribose-5-phosphate, erythrose-4-phosphate. Due to the important impact of metabolic burden on biotechnological processes, several groups have worked on developing strategies to overcome this problem, like reduction of plasmid copy number (Seo JH, Bailey JE. Biotechnol Bioeng 1985;27:1668-1674; Jones KL, Kim S, Keasling JD. Metab Eng 2000;3:328-338), chromosomal insertion of the gene which product is desired, or changing the plasmid-coded antibiotic resistance gene (Hong Y, Pasternak JJ, Glick BR. Can J Microbiol 1995;41:624-628). However, few efforts have been attempted to overcome the reduction of growth rate due to protein over-expression, by modifying central metabolic pathways (Chou C-H, Bennett GN, San KY. Biotechnol Bioeng 1994;44:952-960). We constructed a high-copy number plasmid carrying the gene for glucose-6-phosphate dehydrogenase, zwf, under the control of an inducible trc promoter (pTRzwf04 plasmid). By transforming a wild-type strain and inducing with IPTG, it was possible to recover growth-rate from 0.46 h(-1) (uninduced) to 0.64 h(-1) (induced). The same transformation in an Escherichia coli zwf(-), allows a growth-rate recovery from 0.43 h(-1) (uninduced) to 0.62 h(-1) (induced). We also studied this effect as part of a laboratory-scale biotechnology process: production of a recombinant insulin peptide by co-transforming E. coli JM101 strain with pTRzwf07, a low-copy-number plasmid that carries the same inducible construction as pTRzwf04, and with the pTEXP-MMRPI vector that carries a TrpLE-proinsulin hybrid gene. In this system, production of TrpLE-proinsulin strongly reduces growth rate; however, overexpression of zwf gene recovers with a growth rate from 0.1 h(-1) in the TrpLE-proinsulin induced strain, to 0.37 h(-1) when both zwf and TrpLE-proinsulin genes were induced. In this paper, we show that the engineering of the pentose phosphate pathway by modulation of the zwf gene expression level partially overcomes the possible bottleneck for the supply of building blocks and reducing power synthesized through the PP pathway, that are required for plasmid replication and plasmid-encoded protein expression.

Cell Proliferation↗

PubMed2004

Role of pyruvate oxidase in Escherichia coli strains lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system.

We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (mu) in glucose minimal medium of 0.1 h(-1) is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a mu of 0.70 h(-1)). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a mu of 0.40 h(-1). In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB(-)derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS(-) strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.

Acetic Acid↗

PubMed2004

Dental emergency rates at two expeditionary medical support facilities supporting operations enduring and Iraqi Freedom.

This study reports dental emergency rates and distribution of causes of dental emergencies at two expeditionary medical support facilities supporting operations Enduring Freedom/ Iraqi Freedom. A retrospective cohort analysis of 9948 soldiers deployed to Prince Sultan Air Base, Kingdom of Saudi Arabia, and 1467 soldiers at Baghdad International Airport, Iraq, was accomplished from a phased deployment from January 2003 to September 2003. Procedures were divided into 11 categories: endodontic, extraction of teeth other than third molars, extraction of third molar teeth, restoration of teeth (caries), restoration of broken teeth (not caries), orthodontic bracket/wire problem, sensitive teeth, temperomandibular pain, periodontal, oral pathology, and prosthodontic. The dental emergency rates for Prince Sultan Air Base and Baghdad International Airport were 153 and 145 dental emergencies per 1000 soldiers per year, respectively. Most of the emergencies were because of dental caries. Pain from third molars was the second most common reason for visiting the dental clinic.

Dental Service, Hospital↗

PubMed2003

Dentistry and bioterrorism.

Dentistry's role in responding to bioterrorism and other catastrophic events is evolving and may involve a wide range of activities. Organized dentistry. local dental societies. and interested individuals should make local emergency response planners aware of the services the dental profession can provide and should work to integrate dental resources to strengthen the disaster response capacity of community health care systems. With effective planning, education, and training, dentists can play a significant role in responding to acts of bioterrorism or other unforeseen events.

Biological Warfare↗