PorA represents the major cell wall channel of the Gram-positive bacterium Corynebacterium glutamicum

Abstract

The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA. porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032deltaporA. Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA.

FIG. 1.

Southern blot analysis of C. glutamicum wild-type ATCC 13032 and C. glutamicum ATCC 13032ΔporA cells. (A) Chromosomal DNA from wild-type cells (wt) and the ΔporA mutant strain (ΔporA) were digested with restriction enzyme BglI, subjected to agarose gel electrophoresis, transferred onto a nylon membrane, and probed with a digoxigenin-labeled oligonucleotide corresponding to the last 33 bp of the porA gene. (B) Schematic representation from the 169-bp fragment released in the chromosome of the wild-type C. glutamicum strain after cleavage with BglI. Dig, digoxigenin

FIG. 2.

(A) Agarose (0.8%) gel from PCR. DNA from C. glutamicum wild-type and ΔporA mutant strains were used as templates for PCR performed with primers Por3 to Por6 (Table 1) for the 1,800-bp fragment of the whole flanking region of porA. The PCR products were electrophoresed on a 0.8% agarose gel. Lane 1, 1-kb ladder; lane 2, PCR product obtained by using wild-type DNA; lane 3, PCR product obtained by using DNA from the ΔporA mutant strain. Note that the PCR product obtained with the ΔporA mutant DNA (lane 3) is smaller, indicating that there was an approximately 150-bp deletion. (B) Alignment of the deletion PCR product with the wild-type sequence. To see how many base pairs were deleted by homologous recombination, the PCR product from lane 3 of the gel was used for sequencing. The missing base pairs are shown without alignment. The boldface type indicates the three open reading frames (the numbers above the sequences are accession numbers). The GenBank accession number of the genome of C. glutamicum ATCC 13032 is NC_003450. Proteins smaller than 60 amino acids (aa) were excluded from the sequence when it was reviewed. This is the reason why porA and the 58-amino-acid protein upstream have just one accession number, whereas the polyphosphate kinase has two accession numbers, one from the first submission and the second from the reviewed submission (in parentheses). As the data show, the homologous recombination did not affect any other protein; it affected only the entire porA gene and some base pairs from the intergenic regions up- and downstream. The numbers before the chromosomal DNA from the GenBank are the positions in the whole genome. (C) RT of total mRNA from wild-type C. glutamicum and the ΔporA mutant strain. Total mRNA was converted into cDNA and amplified with the porA-specific primers Por1 and Por2 (Table 1). Lane 1, 100-bp ladder: lane 2, PCR amplification of the wild-type strain; lane 3, PCR amplification of the ΔporA mutant. Note that no amplification product was detected for the deletion mutant (lane 3).

FIG. 3.

Growth curves for wild-type C. glutamicum and the ΔporA mutant shown on a semilogarithmic scale. (A) C. glutamicum wild type (WT) and ΔporA mutant grown in rich medium (BHI medium) for about 9 h at 30°C. (B) C. glutamicum and ΔporA mutant grown in minimal medium for about 9 h at 30°C.

FIG. 4.

Single-channel recordings of diphytanoylphosphatidylcholine-phosphatidylserine (molar ratio, 4:1)-n-decane membranes in the presence of 5 μg of a chloroform methanol extract of whole C. glutamicum wild-type cells per ml precipitated with ether and dissolved in 0.4% LDAO-10 mM Tris-HCl (pH 8) (A) and 250 μg of the chloroform methanol extract of whole C. glutamicum ΔporA mutant cells per ml precipitated with ether and dissolved in 0.4% LDAO-10 mM Tris-HCl (pH 8). The aqueous phase contained unbuffered 1 M KCl (pH 6). The applied voltage was 20 mV, and the temperature was 20°C.

FIG. 5.

Single-channel recording of a diphytanoylphosphatidylcholine-phosphatidylserine (molar ratio, 4:1)-n-decane membrane in the presence of 100 ng of synthetic PorA per ml dissolved in 0.4% LDAO-10 mM Tris-HCl (pH 8). The aqueous phase contained 1 M unbuffered KCl (pH 6). The applied voltage was 20 mV, and the temperature was 20°C.