Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis.

Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake (3)H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis.

The repABC Plasmids with Quorum-Regulated Transfer Systems in Members of the Rhizobiales Divide into Two Structurally and Separately Evolving Groups.

The large repABC plasmids of the order Rhizobiales with Class I quorum-regulated conjugative transfer systems often define the nature of the bacterium that harbors them. These otherwise diverse plasmids contain a core of highly conserved genes for replication and conjugation raising the question of their evolutionary relationships. In an analysis of 18 such plasmids these elements fall into two organizational classes, Group I and Group II, based on the sites at which cargo DNA is located. Cladograms constructed from proteins of the transfer and quorum-sensing components indicated that those of the Group I plasmids, while coevolving, have diverged from those coevolving proteins of the Group II plasmids. Moreover, within these groups the phylogenies of the proteins usually occupy similar, if not identical, tree topologies. Remarkably, such relationships were not seen among proteins of the replication system; although RepA and RepB coevolve, RepC does not. Nor do the replication proteins coevolve with the proteins of the transfer and quorum-sensing systems. Functional analysis was mostly consistent with phylogenies. TraR activated promoters from plasmids within its group, but not between groups and dimerized with TraR proteins from within but not between groups. However, oriT sequences, which are highly conserved, were processed by the transfer system of plasmids regardless of group. We conclude that these plasmids diverged into two classes based on the locations at which cargo DNA is inserted, that the quorum-sensing and transfer functions are coevolving within but not between the two groups, and that this divergent evolution extends to function.

The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism.

UNLABELLED: The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. IMPORTANCE: Our findings show that Francisella novicida has evolved two functional biotin protein ligases, BplA and BirA. BplA is a much more efficient enzyme than BirA, and its expression is significantly induced upon infection of macrophages. Only BplA is required for F. novicida pathogenicity, whereas BirA prevents wasteful biotin synthesis. These data demonstrate that the atypical occurrence of two biotin protein ligases in F. novicida is linked to distinct roles in virulence and biotin metabolism.

A series of medium and high copy number arabinose-inducible Escherichia coli expression vectors compatible with pBR322 and pACYC184.

The original pBAD24 plasmid and the derived lower copy number (the pBAD322 series) expression vectors have been widely used in Escherichia coli, Salmonella enterica, and related bacteria. However, a flexible pBAD expression system has been available only in pMB1 (ColE1) vectors. We report a series of pBAD vectors that replicate using the origin of plasmid RSF1030 that are compatible with pMB1 (ColE1) and p15A (pACYC) vectors. Both high (≥pBAD24) and medium (~pBAD322) copy number plasmids encoding resistance to ampicillin, chloramphenicol, kanamycin, tetracycline, spectinomycin/streptomycin, gentamycin, or trimethoprim are available.

The wing of a winged helix-turn-helix transcription factor organizes the active site of BirA, a bifunctional repressor/ligase.

The BirA biotin protein ligase of Escherichia coli belongs to the winged helix-turn-helix (wHTH) family of transcriptional regulators. The N-terminal BirA domain is required for both transcriptional regulation of biotin synthesis and biotin protein ligase activity. We addressed the structural and functional role of the wing of the wHTH motif in both BirA functions. A panel of N-terminal deletion mutant proteins including a discrete deletion of the wing motif were unable to bind DNA. However, all the N-terminal deletion mutants weakly complemented growth of a ΔbirA strain at low biotin concentrations, indicating compromised ligase activity. A wing domain chimera was constructed by replacing the BirA wing with the nearly isosteric wing of the E. coli OmpR transcription factor. Although this chimera BirA was defective in operator binding, it was much more efficient in complementation of a ΔbirA strain than was the wing-less protein. The enzymatic activities of the wing deletion and chimera proteins in the in vitro synthesis of biotinoyl-5'-AMP differed greatly. The wing deletion BirA accumulated an off pathway compound, ADP, whereas the chimera protein did not. Finally, we report that a single residue alteration in the wing bypasses the deleterious effects caused by mutations in the biotin-binding loop of the ligase active site. We believe that the role of the wing in the BirA enzymatic reaction is to orient the active site and thereby protect biotinoyl-5'-AMP from attack by solvent. This is the first evidence that the wing domain of a wHTH protein can play an important role in enzymatic activity.

Remarkable diversity in the enzymes catalyzing the last step in synthesis of the pimelate moiety of biotin.

Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo. Moreover, purified BioG proteins have esterase activity against pimeloyl-ACP methyl ester, the physiological substrate of BioH. Two of the BioG proteins block biotin synthesis when highly expressed and these toxic proteins were shown to have more promiscuous substrate specificities than the non-toxic BioG proteins. A postulated BioG-BioC fusion protein was shown to functionally replace both the BioH and BioC functions of E. coli. Although the BioH, BioG and BioK esterases catalyze a common reaction, the proteins are evolutionarily distinct.

Altered regulation of Escherichia coli biotin biosynthesis in BirA superrepressor mutant strains.

Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5'-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues located close to the dimerization interface of BirA. However, two mutations were located at sites well removed from the interface. The properties of the superrepressor mutants strengthen and extend other data indicating that BirA function entails extensive interactions among the three domains of the protein and show that normal ligase activity does not ensure normal DNA binding.

Quorum-sensing signal production by Agrobacterium vitis strains and their tumor-inducing and tartrate-catabolic plasmids.

Agrobacterium vitis strains, their tumor-inducing (pTi) and tartrate utilization (pTr) plasmid transconjugants and grapevine tumors were analyzed for the presence of N-acyl-homoserine lactones (AHLs). All wild-type A. vitis strains produced long-chain signals. PCR analysis of the A. vitis long-chain AHL synthase gene, avsI, showed the predicted amplicon. Agrobacterium tumefaciens UBAPF2 harboring various A. vitis pTi plasmids produced N-(3-oxo-octanoyl)-l-homoserine lactone encoded also by pTis of A. tumefaciens. UBAPF2 transconjugants carrying pTrs except for pTrTm4 and pTrAB3, also produced an AHL. UBAPF2 transconjugants carrying pTrAT6, pTrAB4 and pTrRr4 or pTiNi1 produced two additional AHLs not observed in the corresponding wild-type strains. We also provide evidence for in situ production of AHLs in grapevine crown gall tumors of greenhouse and field origin.