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A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

ABSTRACT: The signal sequence of the murine serine protease inhibitor PAI-2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on export mediated by wild-type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppress the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI-2 signal sequence. Using a malE signal sequence mutant, which has a Mal-phenotype in secG mutant strains, we have isolated extragenic Mal+ suppressors. Most suppressors map to secY, and several are allele-specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of functional translocation complexes: with or without SecG.

SUBMITTER: Bost S

PROVIDER: S-EPMC394532 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

Bost S S Belin D D

The EMBO journal 19950901 18

The signal sequence of the murine serine protease inhibitor PAI-2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino ...[more]

PMID: 7556084

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Project description:BACKGROUND:Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T). RESULTS:Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (?speB, ?speC, ?speF, ?speB/C and ?speD/E), and in polyamines transport systems (?potE, ?yeeF, ?potABCD and ?potFGHI). Contrary to what was observed for MG1655, the ?potE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ?speB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ?speB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed. CONCLUSIONS:Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

Project description:Escherichia coli are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic E. coli (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental ("dairy-farm" E. coli [DFEC]) strains, we found that only the fec locus (fecIRABCDE) for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes (P < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the fecA gene and of the inner membrane transport component fecB gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the fec system was shown to affect the ability of E. coli to grow in milk. While the rate of growth in milk of fec-positive (fec+) DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking fec Furthermore, deletion of fec reduced the rate of growth in milk of MPEC strain P4, whereas fec-transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of fec in E. coli intramammary pathogenicity was investigated in vivo in cows, with results showing that an MPEC P4 mutant lacking fec lost its ability to induce mastitis, whereas the fec+ DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity.IMPORTANCE Bovine mastitis is the major infectious disease in dairy cows and the leading cause of economic loss to the global dairy industry, directly contributing to the price of dairy products on supermarket shelves and the financial hardships suffered by dairy farmers. Mastitis is also the leading reason for the use of antibiotics in dairy farms. Good farm management practices in many countries have dramatically reduced the incidence of contagious mastitis; however, the problems associated with the incidence of environmental mastitis caused by bacteria such as Escherichia coli have proven intractable. E. coli bacteria cause acute mastitis, which affects the health and welfare of cows and in extreme cases may be fatal. Here we show for the first time that the pathogenicity of E. coli causing mastitis in cows is highly dependent on the fecIRABCDE ferric citrate uptake system that allows the bacterium to capture iron from citrate. The Fec system is highly expressed during infection in the bovine udder and is ubiquitous in and necessary for the E. coli bacteria that cause mammary infections in cattle. These results have far-reaching implications, raising the possibility that mastitis may be controllable by targeting this system.

Dataset Information

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

Publications

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

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