Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family

Abstract

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in alpha-proteobacteria but not in other prokaryotes analyzed.

FIG. 1.

Peptide-sensitive growth of Tn mutants. (A) Growth of Tn mutants 1-75 (circles) and 1-8 (triangles) compared to the control strain, C. glutamicum ATCC 14752/pZ1(diamonds), in the presence (solid symbols) or absence (open symbols) of 3 mM Ile-Ile. (B) Growth of the deletion mutant 13032ΔbrnFE with control vector pJC1 (triangles) or pJC1brnFE (circles) compared to the wild-type 13032/pZ1 (diamonds) in the presence (solid symbols) or absence (open symbols) of 5 mM Ile-Ile.

FIG. 2.

Overview on the brnE-brnF-lrp locus of C. glutamicum and regions with identified promoter activity. In the upper part of the figure, the thick arrows represent the genes with their sizes in base pairs. Above them, two fragments used for plasmid construction and lrp inactivation, respectively, are given, as well as the part of the genome deleted in the brnFE deletion mutant. In the lower part of the figure, the two fragments, Pro1 and Pro2, used for primer extension are depicted, together with the sequence of the promoters identified. The transcriptional start sites are marked “+1.” These are underlined, as are the −10 and −35 promoter regions. The initiation codons for lrp and brnF are indicated in boldface. The separate brnE-transcript initiation might not contribute for polypeptide formation (see the text).

FIG. 3.

Mapping of the transcriptional start sites of lrp (plrp), brnF (pbrnF), and brnE (pbrnE) by primer extension analysis. In each experiment, the primer extension product was run in the numbered lane. The sequencing ladder (labeled A, C, G, and T) of the coding strand was generated by using the same primer that was used for primer extension. The respective transcriptional start site is indicated by the arrow.

FIG. 4.

Internal l-isoleucine concentrations and isoleucine efflux after brnFE expression. (A) Efflux (solid symbols) and internal l-isoleucine concentrations (open symbols) upon addition of 3 mM Ile-Ile to 13032/pJC1brnFE (squares) and 13032ΔbrnFE/pJC1 (triangles) compared to the wild-type strain 13032/pZ1 (diamonds). (B) Same as panel A, except that 10 mM Ile-Ile was used with strain 13032/pJC1brnFE and 0.5 mM Ile-Ile was used with strain 13032ΔbrnFE/pJC1.

FIG. 5.

Efflux (solid symbols) and internal concentrations (open symbols) of l-valine and l-leucine by C. glutamicum ATCC 13032ΔbrnFE/pJC1 (triangles) and ATCC 13032ΔbrnFE/pJC1brnFE (squares). (A) Response in the presence of 5 mM Ala-Val. (B) Response in the presence of 5 mM Leu-Leu.

FIG. 6.

Efflux (solid symbols) and internal concentrations (open symbols) of l-leucine (squares) and l-isoleucine (diamonds) with C. glutamicum ATCC 13032 after the addition of 10 mM Leu-Ile.

FIG. 7.

Phylogenetic trees for the BrnF family (A) and the BrnE family (B). Protein abbreviations are as defined in Table 3. The trees were based on multiple alignments generated by using the CLUSTAL X program (36).

FIG. 8.

Average hydropathy (solid line), amphipathicity (shaded line), and similarity (dashed line) for the BrnF and BrnE families. The plots were generated by using the AveHAS program (43), based on multiple alignments generated with the CLUSTAL X program.