The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.

/pdf/experimental-determination-and-system-level-analysis-of-2cqosre9sm.pdf

Experimental Determination and System Level Analysis of Essential Genes in Escherichia coli MG1655

Development of genetic circuitry exhibiting toggle switch or oscillatory behavior in Escherichia coli.

Technologies that can provide a cell-wide perspective are very useful. Important “global” technologies using two-dimensional methods were pioneered by O'Farrell and coworkers (O'Farrell 1975) for protein analysis (proteomics), and by Fodor and coworkers (Fodor et al. 1993) for nucleic acid analysis (genomics). These technologies and subsequent refinements allow for global analysis of the important macromolecules of cells that convey the information flow from DNA to RNA to protein. However, the information initially encoded in the genome is ultimately displayed at the cellular level as cellular traits or phenotypes. This paper describes a new technology called Phenotype MicroArrays (PM) that provides an analogous two-dimensional array technology for analysis of live cells (phenomics) to measure hundreds or thousands of cellular properties simultaneously.

A technology for global analysis of cellular phenotypes was first proposed by Bochner (1989b) using microplates for high-throughput assays. Two groups working in genomics of Saccharomyces cerevisiae as a model system recently have tested a large number of strains against 96 (Ross-Macdonald et al. 1999) or 288 (Reiger et al. 1997, 1999) growth phenotypes. These groups used microplate technology to test the growth of yeast strains on the surface of agar. A problem with this approach is that it is difficult to scale it efficiently for high-throughput testing. Agar media have a short shelf life and must be prepared freshly. Scoring of growth is rather subjectively and inefficiently performed with daily visual or photographic records. Estimation of apparent cell mass on a surface is difficult and can be misleading. For example, cellular changes can cause colonies to spread or secrete capsular polysaccharide, thereby appearing much larger than the actual cell number. An ideal high-throughput system would allow for automated, kinetic reading and storing of quantitative phenotypic data directly into computer databases amenable to bioinformatics analyses.

At Biolog, we have employed testing of cellular phenotypes using cell respiration as a reporter system since 1984 (Bochner 1988, 1989a,b). The assay chemistry uses a tetrazolium dye, usually tetrazolium violet, to colorimetrically detect the respiration of cells. Reduction of this dye results in formation of a purple color and because the dye reduction is essentially irreversible under physiological conditions, it accumulates in the well over a period of hours, amplifying the signal and integrating the amount of breathing over time. This provides several major benefits: (1) The color change is easy to monitor; (2) the color change is easy to quantitate; (3) the color change is very sensitive and highly reproducible; and (4) cell respiration can occur independent of cell growth and, in some cases, can measure phenotypes that do not lead to growth.

As part of this technology, the OmniLog instrument has been developed for the purpose of reading and recording the color change in PM assays. The instrument cycles microplates in front of a color CCD camera to read 50 in as little as 5 min and provides quantitative and kinetic information about the response of cells in the PMs. Data are stored directly into computer files and can be recalled and compared with other data at any time.

Figure ​Figure11 shows how cell respiration can be coupled to a large number and a wide range of cellular phenotypes. In a normal growth situation a coordinated sequence of events must occur. Cells must transport nutrients, catabolize and reform them, produce essential small molecule components, polymerize these into macromolecules, create and assemble subcellular structures, etc. If all of these processes are working normally, the cell can grow and there will be an actual physical flow of electrons from the carbon source to NADH, down the electron transport chain of the cell, and, ultimately, onto the tetrazolium dye to produce the purple color. If one of these processes is working at a subnormal rate it will become a pinchpoint, restricting this flow and resulting in a decrease in purple color. The severity of the restriction is reflected in the degree of loss of purple color. Total loss of function will result in no growth and no purple color. Therefore, colorimetric assay of respiration can provide a virtually universal reporter system for phenomic testing. 



Figure 1

Respiration pathways coupled to cell physiology.



About half of the genes from genomic sequencing efforts have no ascribed function, and even genes with ascribed functions are based primarily on DNA sequence homology, with little or no direct experimental data. Large-scale gene knock-out projects for S. cerevisiae (Burns et al. 1994) have been essentially completed and other projects from Bacillus subtilis (Vagner et al. 1998) to E. coli (Link et al. 1997; Datsenko and Wanner 2000; F. Blattner, pers. comm.) to mouse (Zambrowicz et al. 1998) are at various stages. We developed the PM technology anticipating that it can be used to analyze the effects of loss of gene function, providing a direct experimental linkage between genotype and phenotype. The idea is simply to compare isogenic pairs of strains in PMs to directly assay for the cellular effects of loss of gene function.

/pdf/phenotype-microarrays-for-high-throughput-phenotypic-testing-jjt9835pc8.pdf

Phenotype MicroArrays for High-Throughput Phenotypic Testing and Assay of Gene Function

https://folk.ntnu.no/drablos/papers/24_DNA_Repair_2004.pdf

Alkylation damage in DNA and RNA--repair mechanisms and medical significance.

ς54 has several features that distinguish it from other sigma factors in Escherichia coli: it is not homologous to other ς subunits, ς54-dependent expression absolutely requires an activator, and the activator binding sites can be far from the transcription start site. A rationale for these properties has not been readily apparent, in part because of an inability to assign a common physiological function for ς54-dependent genes. Surveys of ς54-dependent genes from a variety of organisms suggest that the products of these genes are often involved in nitrogen assimilation; however, many are not. Such broad surveys inevitably remove the ς54-dependent genes from a potentially coherent metabolic context. To address this concern, we consider the function and metabolic context of ς54-dependent genes primarily from a single organism, Escherichia coli, in which a reasonably complete list of ς54-dependent genes has been identified by computer analysis combined with a DNA microarray analysis of nitrogen limitation-induced genes. E. coli appears to have approximately 30 ς54-dependent operons, and about half are involved in nitrogen assimilation and metabolism. A possible physiological relationship between ς54-dependent genes may be based on the fact that nitrogen assimilation consumes energy and intermediates of central metabolism. The products of the ς54-dependent genes that are not involved in nitrogen metabolism may prevent depletion of metabolites and energy resources in certain environments or partially neutralize adverse conditions. Such a relationship may limit the number of physiological themes of ς54-dependent genes within a single organism and may partially account for the unique features of ς54 and ς54-dependent gene expression.

https://mmbr.asm.org/content/mmbr/65/3/422.full.pdf

Metabolic Context and Possible Physiological Themes of ς54-Dependent Genes in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.

Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

Escherichia coli is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential ς54-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the ς54-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced 14CO2 from minimal medium containing [14C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated 14CO2 production from [14C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to xdhA. The xdhA mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.

Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage.

Nitrogen limitation induces the nitrogen-regulated (Ntr) response, which includes proteins that assimilate ammonia and scavenge nitrogen. Nitrogen limitation also induces catabolic pathways that degrade four metabolically related compounds: putrescine, arginine, ornithine, and γ-aminobutyrate (GABA). We analyzed the structure, function, and regulation of the gab operon, whose products degrade GABA, a proposed intermediate in putrescine catabolism. We showed that the gabDTPC gene cluster constitutes an operon based partially on coregulation of GabT and GabD activities and the polarity of an insertion in gabT on gabC. A ΔgabDT mutant grew normally on all of the nitrogen sources tested except GABA. The unexpected growth with putrescine resulted from specific induction of gab-independent enzymes. Nac was required for gab transcription in vivo and in vitro. Ntr induction did not require GABA, but various nitrogen sources did not induce enzyme activity equally. A gabC (formerly ygaE) mutant grew faster with GABA and had elevated levels of gab operon products, which suggests that GabC is a repressor. GabC is proposed to reduce nitrogen source-specific modulation of expression. Unlike a wild-type strain, a gabC mutant utilized GABA as a carbon source and such growth required σS. Previous studies showing σS-dependent gab expression in stationary phase involved gabC mutants, which suggests that such expression does not occur in wild-type strains. The seemingly narrow catabolic function of the gab operon is contrasted with the nonspecific (nitrogen source-independent) induction. We propose that the gab operon and the Ntr response itself contribute to putrescine and polyamine homeostasis.

The Escherichia coli gabDTPC Operon: Specific γ-Aminobutyrate Catabolism and Nonspecific Induction

Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages.

Barbara L. Schneider

Papers

Metabolic Context and Possible Physiological Themes of ς54-Dependent Genes in Escherichia coli

Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage.

The Escherichia coli gabDTPC Operon: Specific γ-Aminobutyrate Catabolism and Nonspecific Induction

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica serovar Typhimurium