Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation.

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis.

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells' area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.

Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation.

Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram-negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C-terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.

Novel Titanocene Y derivative with albumin affinity exhibits improved anticancer activity against platinum resistant cells.

The antitumor activity of Ti(IV)-based compounds put them in the spotlight for cancer treatment in the past, but their lack of stability in vivo due to a high rate of hydrolysis has hindered their development as antitumor drugs. As a possible solution for this problem, we have reported a synthesis strategy through which we combined a titanocene fragment, a tridentate ligand, and a long aliphatic chain. This strategy allowed us to generate a titanium compound (Myr-Ti) capable of interacting with albumin, highly stable in water and with cytotoxic activity in tumor cells[1]. Following a similar strategy, now we report the synthesis of a new compound (Myr-TiY) derived from titanocene Y that shows antitumoral activity in a cisplatin resistant model with a 50% inhibitory concentration (IC50) of 41-76 µM. This new compound shows high stability and a strong interaction with human serum albumin. Myr-TiY has a significant antiproliferative and proapoptotic effect on the tested cancer cells and shows potential tumor selectivity when assayed in non-tumor human epithelial cells being more selective (1.3-3.8 times) for tumor cells than cisplatin. These results lead us to think that the described synthesis strategy could be useful to generate compounds for the treatment of both cisplatin-sensitive and cisplatin-resistant cancers.

Needle length control and the secretion substrate specificity switch are only loosely coupled in the type III secretion apparatus of Shigella.

The type III secretion apparatus (T3SA), which is evolutionarily and structurally related to the bacterial flagellar hook basal body, is a key virulence factor used by many gram-negative bacteria to inject effector proteins into host cells. A hollow extracellular needle forms the injection conduit of the T3SA. Its length is tightly controlled to match specific structures at the bacterial and host-cell surfaces but how this occurs remains incompletely understood. The needle is topped by a tip complex, which senses the host cell and inserts as a translocation pore in the host membrane when secretion is activated. The interaction of two conserved proteins, inner-membrane Spa40 and secreted Spa32, respectively, in Shigella, is proposed to regulate needle length and to flick a type III secretion substrate specificity switch from needle components/Spa32 to translocator/effector substrates. We found that, as in T3SAs from other species, substitution N257A within the conserved cytoplasmic NPTH region in Spa40 prevented its autocleavage and substrate specificity switching. Yet, the spa40(N257A) mutant made only slightly longer needles with a few needle tip complexes, although it could not form translocation pores. On the other hand, Δspa32, which makes extremely long needles and also formed only few tip complexes, could still form some translocation pores, indicating that it could switch substrate specificity to some extent. Therefore, loss of needle length control and defects in secretion specificity switching are not tightly coupled in either a Δspa32 mutant or a spa40(N257A) mutant.

Comparative analysis of EspF variants in inhibition of Escherichia coli phagocytosis by macrophages and inhibition of E. coli translocation through human- and bovine-derived M cells.

The EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspF(O127) is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspF(O127) has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of different espF alleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. The espF gene from E. coli serotype O157 (espF(O157)) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell coculture system in comparison to espF(O127) and espF(O26). In contrast, espF(O157) was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

The Shigella T3SS needle transmits a signal for MxiC release, which controls secretion of effectors.

Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacteria, including animal and plant pathogens. They inject 'effector' proteins through a 'needle' protruding from the bacterial surface directly into eukaryotic cells after assembly of a 'translocator' pore in the host plasma membrane. Secretion is a tightly regulated process, which is blocked until physical contact with a host cell takes place. Host cell sensing occurs through a distal needle 'tip complex' and translocators are secreted before effectors. MxiC, a Shigella T3SS substrate, prevents premature effector secretion. Here, we examine how the different parts of T3SSs work together to allow orderly secretion. We show that T3SS assembly and needle tip composition are not altered in an mxiC mutant. We find that MxiC not only represses effector secretion but that it is also required for translocator release. We provide genetic evidence that MxiC acts downstream of the tip complex and then the needle during secretion activation. Finally, we show that the needle controls MxiC release. Therefore, for the first time, our data allow us to propose a model of secretion activation that goes from the tip complex to cytoplasmic MxiC via the needle.

Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression.

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE(+) and SopE(-) S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH-gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA-gfp reporter mirrored that of PprgH-gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.

New titanocene derivative with improved stability and binding ability to albumin exhibits high anticancer activity.

Titanium-based therapies have emerged as a promising alternative for the treatment of cancer patients, particularly those with cisplatin resistant tumors. Unfortunately, some titanium compounds show stability and solubility problems that have hindered their use in clinical practice. Here, we designed and synthesized a new titanium complex containing a titanocene fragment, a tridentate ligand to improve its stability in water, and a long aliphatic chain, designed to facilitate a non-covalent interaction with albumin, the most abundant protein in human serum. The stability and human serum albumin affinity of the resulting titanium complex was investigated by UV-Vis absorption and fluorescence spectroscopy techniques. Complex [TiCp2{(OOC)2py-O-myr}] (3) (myr = C14H29, py = pyridine) and its analogous [TiCp2{(OOC)2py-OH}] (4), lacking the aliphatic chain, showed improved stability in phosphate saline buffer compared with [TiCp2Cl2] (1). 3 showed a strong interaction with human serum albumin in a 1:1 stoichiometry. The cytotoxic effect of 3 was higher compared to [TiCp2Cl2] in tumor cell lines and showed potential tumor selectivity when assayed in non-tumor human epithelial cells. Finally, 3 showed an antiproliferative effect on cancer cells, decreasing the population in the S phase, and increasing apoptotic cells in a significant manner. All this makes the novel Ti(IV) compound 3 a firm candidate to continue further studies of its therapeutic potential in vitro and in vivo.

The extreme C terminus of Shigella flexneri IpaB is required for regulation of type III secretion, needle tip composition, and binding.

Type III secretion systems (T3SSs) are widely distributed virulence determinants of Gram-negative bacteria. They translocate bacterial proteins into host cells to manipulate them during infection. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region, and a hollow needle protruding from the bacterial surface. The distal tip of mature, quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. However, its mode of needle association is unknown. Here, we show that deletion of 3 or 9 residues at the C terminus of IpaB leads to fast constitutive secretion of late effectors, as observed in a DeltaipaB strain. Like the DeltaipaB mutant, mutants with C-terminal mutations also display hyperadhesion. However, unlike the DeltaipaB mutant, they are still invasive and able to lyse the internalization vacuole with nearly wild-type efficiency. Finally, the mutant proteins show decreased association with needles and increased recruitment of IpaC. Taken together, these data support the notion that the state of the tip complex regulates secretion. We propose a model where the quiescent needle tip has an "off" conformation that turns "on" upon host cell contact. Our mutants may adopt a partially "on" conformation that activates secretion and is capable of recruiting some IpaC to insert pores into host cell membranes and allow invasion.

LuxS-based quorum sensing does not affect the ability of Salmonella enterica serovar Typhimurium to express the SPI-1 type 3 secretion system, induce membrane ruffles, or invade epithelial cells.

Bacterial species can communicate by producing and sensing small autoinducer molecules by a process known as quorum sensing. Salmonella enterica produces autoinducer 2 (AI-2) via the luxS synthase gene, which is used by some bacterial pathogens to coordinate virulence gene expression with population density. We investigated whether the luxS gene might affect the ability of Salmonella enterica serovar Typhimurium to invade epithelial cells. No differences were found between the wild-type strain of S. Typhimurium, SL1344, and its isogenic luxS mutant with respect to the number and morphology of the membrane ruffles induced or their ability to invade epithelial cells. The dynamics of the ruffling process were also similar in the wild-type strain (SL1344) and the luxS mutant. Furthermore, comparing the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion profiles of wild-type SL1344 and the luxS mutant by Western blotting and measuring the expression of a single-copy green fluorescent protein fusion to the prgH (an essential SPI-1 gene) promoter indicated that SPI-1 expression and activity are similar in the wild-type SL1344 and luxS mutant. Genetic deletion of luxS did not alter the virulence of S. Typhimurium in the mouse model, and therefore, it appears that luxS does not play a significant role in regulating invasion of Salmonella in vitro or in vivo.

Silenced retrotransposons are major rasiRNAs targets in Arabidopsis galls induced by Meloidogyne javanica.

Root-knot nematodes (RKNs, Meloidogyne spp.) are sedentary biotrophic pathogens that establish within the vascular cylinder of plant roots, forming a gall and inducing several feeding cells, giant cells (GCs), essential for completion of their life cycle. GCs suffer gene expression changes, repeated mitosis and endoreduplication events. Transcriptomics has revealed that an extensive down-regulation of transcripts, a molecular signature of early-developing galls and GCs that is conserved in tomato and Arabidopsis, may be achieved through small RNA (sRNA) gene silencing pathways. The role of some microRNAs (miRNAs) in plant-RKN interactions has recently been addressed, but little is known about the regulatory roles of other sRNA types. Here, we perform a differential accumulation analysis to show which repeat-associated small interfering RNAs (rasiRNAs) are distinctive or enriched in early Arabidopsis galls vs. uninfected roots. Those distinctive from galls are preferentially located in pericentromeric regions with predominant sizes of 24 and 22 nucleotides. Gall-distinctive rasiRNAs target primarily GYPSY and COPIA retrotransposons, which show a marked repression in galls vs. uninfected roots. Infection tests and phenotypic studies of galls from Meloidogyne javanica in Arabidopsis mutants impaired in post-transcriptional gene silencing and/or canonical RNA-directed DNA methylation (RdDM) pathways, as well as quantitative polymerase chain reaction analysis, suggest the implication of canonical and non-canonical RdDM pathways during gall formation, possibly through the regulation of retrotransposons. This process may be crucial for the maintenance of genome integrity during the reprogramming process of galls/GCs from their vascular precursor cells, and/or to ensure a faithful DNA replication during the repeated mitosis/endoreduplication that concurs with feeding site formation.

Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility.

Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB((P28T)) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB((P28T)) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the F(O)F(1)-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.

GFP plasmid-induced defects in Salmonella invasion depend on plasmid architecture, not protein expression.

We have investigated the impact of plasmids and GFP expression on invasion of cultured epithelial cells by Salmonella enterica Typhimurium strain SL1344. The invasiveness of SL1344 carrying plasmids derived from pBR322, encoding promoterless GFP or constitutively expressed rpsM-GFP, was compared under optimal growth conditions with that of SL1344(pBR322), unmodified SL1344 and a strain with chromosome-integrated rpsM-GFP. The strain carrying pBR322 exhibited normal invasion, but the presence of modified plasmids impaired invasiveness, and impairment was exacerbated by plasmid-encoded chloramphenicol resistance (CmR). Using a different antibiotic resistance marker, kanamycin (KmR), did not impair invasiveness. Despite the effect of plasmid-encoded CmR, the strain containing chromosomally encoded GFP, also carrying a CmR gene, was as invasive as the wild-type. To investigate the mechanism by which plasmid carriage decreases invasion, we monitored SPI-1 gene expression using prgH promoter activity as an index of SPI-1 activity. An SL1344 strain with a chromosome-integrated prgH::gfp reporter construct exhibited lower GFP expression during exponential phase when carrying plasmids incorporating CmR or gfp, mirroring invasion data. These data provide evidence that suppression of SPI-1 gene expression is a major factor in the loss of invasiveness associated with plasmid carriage. Our findings also indicate that some plasmids, especially those carrying CmR, should be used with caution, as virulence traits and gene expression may be affected by their presence. Integration of reporter proteins into the bacterial chromosome, however, appears to circumvent the adverse effects observed with plasmids.

Salmonella translocates across an in vitro M cell model independently of SPI-1 and SPI-2.

We have used an in vitro model of intestinal M cells to examine the mechanisms by which Salmonella enterica translocates across these specialized cells, which constitute a primary site of infection of the mammalian host. S. enterica can invade cultured cells by deploying a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI-1) to translocate effector proteins into the host cell cytoplasm that trigger cellular responses, including prominent cytoskeletal rearrangements. After Salmonella enters the host cell, a second TTSS encoded in SPI-2 modulates intracellular trafficking and enables the bacteria to replicate within a modified vacuolar compartment. Within the host intestine, specialized antigen-sampling M cells, which reside in the epithelium overlying lymphoid tissues in the gut, are a preferential site of Salmonella invasion. The mechanisms of infection of M cells remain poorly defined and it is not known whether either SPI-1 or SPI-2 is required for infection of these cells. To address these questions we have employed an in vitro M cell model involving co-culture of polarized Caco-2 intestinal epithelial cells with Raji B cells. S. enterica serovar Typhimurium translocated across Caco-2/Raji co-cultures to a much greater extent than they cross native Caco-2 cell monolayers. Salmonella translocation was greatly reduced by heat treatment or fixation, suggesting that processes distinct from the sampling of inert particles are the main determinants of bacterial translocation. Translocation across both mono-cultured and co-cultured Caco-2 cells was partially inhibited by treatment with the dynamin inhibitor dynasore, but resistant to EIPA, an inhibitor of macropinocytosis. There was no difference between the abilities of wild-type Salmonella Typhimurium and mutants lacking multiple SPI-1 effectors to translocate across the M cell model, although the SPI-1 effector mutants were somewhat attenuated for translocation across native Caco-2 layers. There was also no difference between wild-type and SPI-2 mutants in M cell translocation. Together these data suggest that that SPI-1 and SPI-2 are dispensable for rapid M cell translocation and that infection at these specialized epithelial sites involves distinctive mechanisms that are not reliably modelled using conventional cell culture infection models.

Translocation of enteropathogenic Escherichia coli across an in vitro M cell model is regulated by its type III secretion system.

Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that utilizes a type III secretion system (TTSS) to modulate diverse host cell processes including cytoskeletal dynamics, tight junction permeability and macrophage phagocytosis. Some EPEC strains exhibit selective tropism for the specialized follicle-associated epithelium (FAE) overlying lymphoid follicles in the gut, which is a major site of uptake of inert particulates and pathogens, but do not translocate from the intestinal lumen in significant numbers. We have investigated the interaction of EPEC with FAE using an established in vitro model of the specialized FAE in which polarized enterocyte-like Caco-2 cells cocultured with the Raji B cell line undergo a phenotypic switch to a form that morphologically and functionally resembles the specialized antigen-transporting M cells found within FAE. Having confirmed that coculture with Raji B cells induces brush border reorganization and enhances particle transport across Caco-2 cells, we investigated translocation of bacteria across the M cell model. While Salmonella translocation was markedly upregulated by Raji coculture, transport of wild-type EPEC occurred at similarly low levels across both native Caco-2 and Caco-2/Raji-cocultured layers. Translocation rates were markedly higher for EPEC strains lacking either functional TTSS or the effector protein EspF. These observations resemble previously reported data on the inhibition of macrophage phagocytosis by EPEC, which has also been reported to be dependent on TTSS and EspF. Furthermore, as with macrophage phagocytosis, enhanced translocation of a TTSS mutant was blocked by wortmannin, implicating inhibition of phosphatidyl inositol 3-kinase-mediated signalling in the regulation of M cell translocation by EPEC.

Role of the H domain of the histidine kinase-like protein NifL in signal transmission.

The NifL protein from Azotobacter vinelandii senses both the redox and fixed nitrogen status to regulate nitrogen fixation by controlling the activity of the transcriptional activator NifA. NifL has a domain architecture similar to that of the cytoplasmic histidine protein kinases. It contains two N-terminal PAS domains and a C-terminal transmitter region containing a conserved histidine residue (H domain) and a nucleotide binding GHKL domain corresponding to the catalytic core of the histidine kinases. Despite these similarities, NifL does not exhibit kinase activity and regulates its partner NifA by direct protein-protein interactions rather than phosphorylation. NifL senses the redox status via a FAD co-factor located within the PAS1 domain and responds to the nitrogen status by interaction with the signal transduction protein GlnK, which binds to the GHKL domain. The ability of NifL to inhibit NifA is antagonized by the binding of 2-oxoglutarate to the N-terminal GAF domain of NifA. In this study we have performed site-directed mutagenesis of the H domain of NifL to examine its role in signal transmission. Our results suggest that this domain plays a major role in transmission of signals perceived by the PAS1 and GHKL domains to ensure that NifL achieves the required conformation necessary to inhibit the 2-oxoglutarate-bound form of NifA. Some of the substitutions discriminate the redox and fixed nitrogen sensing functions of NifL implying that the conformational requirements and/or domain interactions necessary for NifA inhibition differ with respect to the signal input.

PII T-loop mutations affecting signal transduction to NtrB also abolish yeast two-hybrid interactions.

Mutations A49P and Delta47-53 at the T loop of the Escherichia coli GlnB (PII) protein impair regulatory interactions with the two-component sensor regulator NtrB (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schepke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). We show here that these mutations also impair interactions between PII and NtrB in the yeast two-hybrid system, indicating that defects in NtrB regulation closely reflect binding impairment. The reported results underline the strength of two-hybrid assays for analysis of interactions involving the T loop of PII proteins.