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Export of L-Isoleucine from Corynebacterium glutamicum: a Two-Gene-Encoded Member of a New Translocator Family. Nicole Kennerknecht, 2002.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  -proteobacteria but not in other prokaryotes analyzed .
Isolation of Three New Surface Layer Protein Genes (slp) from Lactobacillus brevis ATCC 14869 and Characterization of the Change in Their Expression under Aerated and Anaerobic Conditions. Miia Jakava-Viljanen, 2002.Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869 . Under different growth conditions, L . brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently . Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB . Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S- and R-colony type cells, respectively . Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms . The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region . Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions . slpC was a silent gene under the growth conditions tested . The amino acid contents of all the L . brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible . The interspecies identity of the L . brevis S-layer proteins was mainly restricted to the N-terminal regions of those proteins . Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L . brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known .
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