Laura S. Frost

F factor conjugation is a true type IV secretion system

The F sex factor of Escherichia coli is a paradigm for bacterial conjugation and its transfer (tra) region represents a subset of the type IV secretion system (T4SS) family. The F tra region encodes eight of the 10 highly conserved (core) gene products of T4SS including TraAF (pilin), the TraBF, -KF (secretin-like), -VF (lipoprotein) and TraCF (NTPase), -EF, -LF and TraGF (N-terminal region) which correspond to TrbCP, -IP, -GP, -HP, -EP, -JP, DP and TrbLP, respectively, of the P-type T4SS exemplified by the IncP plasmid RP4. F lacks homologs of TrbBP (NTPase) and TrbFP but contains a cluster of genes encoding proteins essential for F conjugation (TraFF, -HF, -UF, -WF, the C-terminal region of TraGF, and TrbCF) that are hallmarks of F-like T4SS. These extra genes have been implicated in phenotypes that are characteristic of F-like systems including pilus retraction and mating pair stabilization. F-like T4SS systems have been found on many conjugative plasmids and in genetic islands on bacterial chromosomes. Although few systems have been studied in detail, F-like T4SS appear to be involved in the transfer of DNA only whereas P- and I-type systems appear to transport protein or nucleoprotein complexes. This review examines the similarities and differences among the T4SS, especially F- and P-like systems, and summarizes the properties of the F transfer region gene products.

Conjugative DNA metabolism in Gram-negative bacteria

Bacterial conjugation in Gram-negative bacteria is triggered by a signal that connects the relaxosome to the coupling protein (T4CP) and transferosome, a type IV secretion system. The relaxosome, a nucleoprotein complex formed at the origin of transfer (oriT), consists of a relaxase, directed to the nic site by auxiliary DNA-binding proteins. The nic site undergoes cleavage and religation during vegetative growth, but this is converted to a cleavage and unwinding reaction when a competent mating pair has formed. Here, we review the biochemistry of relaxosomes and ponder some of the remaining questions about the nature of the signal that begins the process.

The Physiology and Biochemistry of Pili

Publisher Summary This chapter reviews and discusses the structure and function of pili in light of recent advances employing biochemical, immunological, and genetic approaches. Bacterial “fimbriae” or “pili” are thin (2-1 2 nm diameter) non-flagellar protein filaments found on the surfaces of many types of bacteria. The adhesive properties of pili allow them to bind to other bacteria, bacteriophages, mammalian cells, and inert surfaces. The chapter discusses the classification scheme for pili based on morphology, function, and biochemical properties. It describes three major groups of pilus—namely, conjugative, adhesive, and N-methylphenylalanine (NMePhe) pili. The simplest classification of pili on the basis of function is the division into two broad groups: “conjugative” and “adhesive” pili. It is noted that the biochemical properties help to identify the subpopulations of these pili. The conjugative pili show variable preferences for promoting bacterial mating in liquid or on solid media. Flexible pili generally confer the “universal mating type” and promote mating in liquid media and on solid surfaces equally well, whereas rigid pili are usually associated with “surface preferred” or “surface obligatory” mating types.

The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F‐like plasmids

The effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating‐pair formation in liquid media by the transfer systems of the F‐Iike plasmids pOX38 (F), ColB2 and R100‐1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II‐heptose III region of the LPS (rfaF) whereas R100‐1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating‐pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F‐like plasmids (F, ColB2, R100‐1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.

Coordination of Golgin Tethering and SNARE Assembly GM130 BINDS SYNTAXIN 5 IN A p115-REGULATED MANNER

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.

F conjugation: Back to the beginning

Bacterial conjugation as mediated by the F plasmid has been a topic of study for the past 65 years. Early research focused on events that occur on the cell surface including the pilus and its phages, recipient cell receptors, mating pair formation and its prevention via surface or entry exclusion. This short review is a reminder of the progress made in those days that will hopefully kindle renewed interest in these subjects as we approach a complete understanding of the mechanism of conjugation.

The σE stress response is required for stress-induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress‐induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double‐strand‐break (DSB) repair and requires DinB error‐prone DNA polymerase and the SOS DNA‐damage‐ and RpoS general‐stress responses. We report that the RpoE envelope‐protein‐stress response is also required. In a screen for mutagenesis‐defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σE acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ32, which was postulated to affect mutagenesis. I‐SceI‐induced DSBs alleviated much of the rpoE phenotype, implying that σE promoted DSB formation. Thus, a third stress response and stress input regulate DSB‐repair‐associated stress‐induced mutagenesis. This provides the first report of mutagenesis promoted by σE, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.

FinO is an RNA chaperone that facilitates sense–antisense RNA interactions

The protein FinO represses F‐plasmid conjugative transfer by facilitating interactions between the mRNA of the major F‐plasmid transcriptional activator, TraJ, and an antisense RNA, FinP. FinO is known to bind stem–loop structures in both FinP and traJ RNAs; however, the mechanism by which FinO facilitates sense–antisense pairing is poorly understood. Here we show that FinO acts as an RNA chaperone to promote strand exchange and duplexing between minimal RNA targets derived from FinP. This strongly suggests that FinO may function to destabilize internal secondary structures within FinP and traJ RNAs that would otherwise act as a kinetic trap to sense–antisense pairing. The energy for FinO‐catalyzed base‐pair destabilization does not arise from ATP hydrolysis but appears to be supplied directly from FinO RNA binding free energy. An analysis of the activities of mutants that are specifically deficient in strand exchange but not RNA‐binding activity demonstrates that strand exchange is essential to the ability of FinO to mediate sense–antisense RNA recognition, and that this function also plays a role in repression of conjugation in vivo.

Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

Conjugation is a major mechanism for disseminating genetic information in bacterial populations, but the signal that triggers it is poorly understood in gram-negative bacteria. F-plasmid-mediated conjugation requires TraM, a homotetramer, which binds cooperatively to three binding sites within the origin of transfer. Using in vitro assays, TraM has previously been shown to interact with the coupling protein TraD. Here we present evidence that F conjugation also requires TraM-TraD interactions in vivo. A three-plasmid system was used to select mutations in TraM that are defective for F conjugation but competent for tetramerization and cooperative DNA binding to the traM promoter region. One mutation, K99E, was particularly defective in conjugation and was further characterized by affinity chromatography and coimmunoprecipitation assays that suggested it was defective in interacting with TraD. A C-terminal deletion (S79*, where the asterisk represents a stop codon) and a missense mutation (F121S), which affects tetramerization, also reduced the affinity of TraM for TraD. We propose that the C-terminal region of TraM interacts with TraD, whereas its N-terminal domain is involved in DNA binding. This arrangement of functional domains could in part allow TraM to receive the mating signal generated by donor-recipient contact and transfer it to the relaxosome, thereby triggering DNA transfer.

Regulation of the expression of the traM gene of the F sex factor of Escherichia coli

Conjugative F‐plasmid transfer is mediated by the transfer (tra ) region which encodes nearly 40 genes, 25 of which are essential for this process in Escherichia coli. TraM is required for conjugation and is encoded on a separate operon between the origin of transfer and the traJ gene. The traJ gene product is the positive regulator of transcription of the 30 kb tra operon, the first gene of which is traY. Using primer‐extension assays and immunoblots on the F plasmid itself and its derivatives, we demonstrate that F TraM regulates its own expression from two promoters and that it requires TraY as well as expression of the tra operon for maximal traM transcription. traY is the first gene in the tra operon under the control of the TraJ regulator, which is in turn negatively regulated by the antisense RNA, FinP, and the FinO protein. Thus, a control circuit has been established whereby traM is negatively regulated by the FinOP fertility inhibition system through its repression of TraJ expression, which adversely affects transcription of the traY gene.