Publications

• Identification of the btuCED polypeptides and evidence for their role in vitamin B12 transport in Escherichia coli.

  de Veaux L.C., Clevenson D.S., Bradbeer C., Kadner R.J.

  Passage of vitamin B12 across the outer and cytoplasmic membranes of Escherichia coli occurs in two steps, each involving independent transport systems. Since the vitamin accumulated in btuC or btuD mutants is readily released from the cell by chase or osmotic shock and does not undergo the usual ... >> More

  Passage of vitamin B12 across the outer and cytoplasmic membranes of Escherichia coli occurs in two steps, each involving independent transport systems. Since the vitamin accumulated in btuC or btuD mutants is readily released from the cell by chase or osmotic shock and does not undergo the usual metabolic conversions, the products of these genes might participate in transport across the cytoplasmic membrane. Mutations in btuC and btuD are complemented by recombinant plasmids carrying a 3,410-base-pair HindIII-HincII DNA fragment. Transposon Tn1000 mutagenesis and subcloning defined the location of these two genes and showed that they are separated by approximately 800 base pairs. The polypeptides elicited by this fragment and its derivatives were identified by using a maxicell system. The apparent molecular weight of the btuC product was approximately 26,000, that of the btuD product was 29,000. Both polypeptides were associated with the cell membrane. Transposon insertions in the region between btuC and btuD, as well as those in the two genes, conferred a deficiency in vitamin B12 utilization and transport when they were crossed onto the chromosome. This region, termed btuE, encoded a 22,000-Mr polypeptide and lesser amounts of a 20,000-Mr species. A portion of the BtuE protein was released from maxicells by osmotic shock or spheroplast formation. The relative production of BtuE and BtuD in response to plasmids carrying transposon insertions suggested that the three genes are arranged in an operon in the order btuC-btuE-btuD and that internal promoters exist since polarity was incomplete. Substantial elevation of transport activity was engendered by plasmids carrying the intact btu region, but not when any of the btu genes was disrupted. The btuCED region thus may encode a transport system for passage of vitamin B12 across the cytoplasmic membrane. This system bears similarities to periplasmic binding protein-dependent transport systems, although the putative periplasmic component is not required for its function. << Less

  J Bacteriol 167:920-927(1986) [PubMed] [EuropePMC]

• Nucleotide sequence of the btuCED genes involved in vitamin B12 transport in Escherichia coli and homology with components of periplasmic-binding-protein-dependent transport systems.

  Friedrich M.J., Deveaux L.C., Kadner R.J.

  The products of the btuCED region of the Escherichia coli chromosome participate in the transport of vitamin B12 across the cytoplasmic membrane. The nucleotide sequence of the 3,410-base-pair HindIII-HincII DNA fragment carrying a portion of the himA gene and the entire btuCED region was determin ... >> More

  The products of the btuCED region of the Escherichia coli chromosome participate in the transport of vitamin B12 across the cytoplasmic membrane. The nucleotide sequence of the 3,410-base-pair HindIII-HincII DNA fragment carrying a portion of the himA gene and the entire btuCED region was determined. Comparison of the location of the open reading frames with the gene boundaries defined by transposon insertions allowed the assignment of polypeptide products to gene sequences. The btuC product is a highly nonpolar integral membrane protein of molecular weight 31,683. The distribution of hydrophobic regions suggests the presence of numerous membrane-spanning domains. The btuD product is a relatively polar but membrane-associated polypeptide of Mr 27,088 and contains segments bearing extensive homology to the ATP-binding peripheral membrane constituents of periplasmic binding protein-dependent transport systems. Other regions of this protein are similar to portions of the outer membrane vitamin B12 receptor. The btuE product (Mr 20,474) appears to have a periplasmic location. It has the mean hydropathy of a soluble protein but lacks an obvious signal sequence. The cellular locations and structural and sequence homologies of the Btu polypeptides point to the similarity of these three proteins to components of binding protein-dependent transport systems. However, the dependence on a periplasmic vitamin B12-binding protein has not yet been demonstrated. << Less

  J. Bacteriol. 167:928-934(1986) [PubMed] [EuropePMC]