Targeting early proximal-rod component substrate FlgB to FlhB for flagellar-type III secretion in Salmonella.

The Salmonella flagellar secretion apparatus is a member of the type III secretion (T3S) family of export systems in bacteria. After completion of the flagellar motor structure, the hook-basal body (HBB), the flagellar T3S system undergoes a switch from early to late substrate secretion, which results in the expression and assembly of the external, filament propeller-like structure. In order to characterize early substrate secretion-signals in the flagellar T3S system, the FlgB, and FlgC components of the flagellar rod, which acts as the drive-shaft within the HBB, were subject to deletion mutagenesis to identify regions of these proteins that were important for secretion. The ß-lactamase protein lacking its Sec-dependent secretion signal (Bla) was fused to the C-terminus of FlgB and FlgC and used as a reporter to select for and quantify the secretion of FlgB and FlgC into the periplasm. Secretion of Bla into the periplasm confers resistance to ampicillin. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 of FlgB decreased FlgB secretion levels while deleting amino acid 6 through 14 diminished FlgC secretion levels. Further PCR-directed mutagenesis indicated that amino acid F45 of FlgB was critical for secretion. Single amino acid mutagenesis revealed that all amino acid substitutions at F45 of FlgB position impaired rod assembly, which was due to a defect of FlgB secretion. An equivalent F49 position in FlgC was essential for assembly but not for secretion. This study also revealed that a hydrophobic patch in the cleaved C-terminal domain of FlhB is critical for recognition of FlgB at F45.

Mutations in the C-terminal region of the bacteriophage exclusion protein PglX can selectively inactivate restriction in Salmonella.

Salmonella enterica serovar Typhimurium strain LT2 is protected by two DNA restriction-modification systems (HsdRMS and Mod-Res) and a Type I bacteriophage exclusion (BREX) system (BrxA-L). The LB5000 strain was constructed to inactivate restriction but not methylation in all three systems and has been available for decades (L. R. Bullas and J. I. Ryu, J Bacteriol 156:471-474, 1983, https://doi.org/10.1128/jb.156.1.471-474.1983). However, this strain had been heavily mutagenized and contains hundreds of other mutations, including a few in DNA repair genes. Here, we describe the development of a strain that is only mutated for DNA restriction by the three systems and remains competent for DNA modification. We transferred mutations specific to DNA restriction from LB5000 to a wild-type LT2 background. The hsdR and res mutations affected only restriction in the wild-type background, but the brxC and pglZ mutations for the poorly understood BREX system also reduced modification. Amino acids in an unannotated conserved region of PglX in the BREX system were then randomized. Mutations were identified that specifically affected restriction at 37°C but were found to be temperature sensitive for restriction and methylation when tested at 30°C and 42°C. These mutations in PglX are consistent with a domain that communicates DNA methylation information to other BREX effector proteins. Finally, mutations generated in the specificity domain of PglX may have changed the DNA binding site recognized by the BREX system. IMPORTANCE The restriction system mutants constructed in this study will be useful for cloning DNA and transferring plasmids from other bacterial species into Salmonella. We verified which mutations in strain LB5000 resulted in loss of restriction for each restriction-modification system and the BREX system by moving these mutations to a wild-type Salmonella background. The methylase PglX was then mutagenized, which adds to our knowledge of the BREX system that is found in many bacteria but is not well understood. These PglX mutations affected restriction and methylation at different temperatures, which suggests that the C-terminal region of PglX may coordinate interactions between the methylase and other BREX system proteins.

Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of Salmonella flagellin inside macrophages.

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.

Regulatory Cross Talk between Motility and Interbacterial Communication in Salmonella enterica Serovar Typhimurium.

FliA is a broadly conserved σ factor that directs transcription of genes involved in flagellar motility. We previously identified FliA-transcribed genes in Escherichia coli and Salmonella enterica serovar Typhimurium, and we showed that E. coli FliA transcribes many unstable, noncoding RNAs from intragenic promoters. Here, we show that FliA in S Typhimurium also directs the transcription of large numbers of unstable, noncoding RNAs from intragenic promoters, and we identify two previously unreported FliA-transcribed protein-coding genes. One of these genes, sdiA, encodes a transcription factor that responds to quorum-sensing signals produced by other bacteria. We show that FliA-dependent transcription of sdiA is required for SdiA activity, highlighting a regulatory link between flagellar motility and intercellular communication.IMPORTANCE Initiation of bacterial transcription requires association of a σ factor with the core RNA polymerase to facilitate sequence-specific recognition of promoter elements. FliA is a widely conserved σ factor that directs transcription of genes involved in flagellar motility. We previously showed that Escherichia coli FliA transcribes many unstable, noncoding RNAs from promoters within genes. Here, we demonstrate the same phenomenon in Salmonella Typhimurium. We also show that S Typhimurium FliA directs transcription of the sdiA gene, which encodes a transcription factor that responds to quorum-sensing signals produced by other bacteria. FliA-dependent transcription of sdiA is required for transcriptional control of SdiA target genes, highlighting a regulatory link between flagellar motility and intercellular communication.

Integration of the pSLT Plasmid into the Salmonella Chromosome Results in a Temperature-Sensitive Growth Defect Due to Aberrant DNA Replication.

A mutant of Salmonella enterica serovar Typhimurium was isolated that simultaneously affected two metabolic pathways as follows: NAD metabolism and DNA repair. The mutant was isolated as resistant to a nicotinamide analog and as temperature-sensitive for growth on minimal glucose medium. In this mutant, Salmonella's 94-kb virulence plasmid pSLT had recombined into the chromosome upstream of the NAD salvage pathway gene pncA This insertion blocked most transcription of pncA, which reduced uptake of the nicotinamide analog. The pSLT insertion mutant also exhibited phenotypes associated with induction of the SOS DNA repair system, including an increase in filamentous cells, higher exonuclease III and catalase activities, and derepression of SOS gene expression. Genome sequencing revealed increased read coverage extending out from the site of pSLT insertion. The two pSLT replication origins are likely initiating replication of the chromosome near the normal replication terminus. Too much replication initiation at the wrong site is probably causing the observed growth defects. Accordingly, deletion of both pSLT replication origins restored growth at higher temperatures.IMPORTANCE In studies that insert a second replication origin into the chromosome, both origins are typically active at the same time. In contrast, the integrated pSLT plasmid initiated replication in stationary phase after normal chromosomal replication had finished. The gradient in read coverage extending out from a single site could be a simple but powerful tool for studying replication and detecting chromosomal rearrangements. This technique may be of particular value when a genome has been sequenced for the first time to verify correct assembly.

Communication across the bacterial cell envelope depends on the size of the periplasm.

The cell envelope of gram-negative bacteria, a structure comprising an outer (OM) and an inner (IM) membrane, is essential for life. The OM and the IM are separated by the periplasm, a compartment that contains the peptidoglycan. The OM is tethered to the peptidoglycan via the lipoprotein, Lpp. However, the importance of the envelope's multilayered architecture remains unknown. Here, when we removed physical coupling between the OM and the peptidoglycan, cells lost the ability to sense defects in envelope integrity. Further experiments revealed that the critical parameter for the transmission of stress signals from the envelope to the cytoplasm, where cellular behaviour is controlled, is the IM-to-OM distance. Augmenting this distance by increasing the length of the lipoprotein Lpp destroyed signalling, whereas simultaneously increasing the length of the stress-sensing lipoprotein RcsF restored signalling. Our results demonstrate the physiological importance of the size of the periplasm. They also reveal that strict control over the IM-to-OM distance is required for effective envelope surveillance and protection, suggesting that cellular architecture and the structure of transenvelope protein complexes have been evolutionarily co-optimised for correct function. Similar strategies are likely at play in cellular compartments surrounded by 2 concentric membranes, such as chloroplasts and mitochondria.

Case for the genetic code as a triplet of triplets.

The efficiency of codon translation in vivo is controlled by many factors, including codon context. At a site early in the Salmonella flgM gene, the effects on translation of replacing codons Thr6 and Pro8 of flgM with synonymous alternates produced a 600-fold range in FlgM activity. Synonymous changes at Thr6 and Leu9 resulted in a twofold range in FlgM activity. The level of FlgM activity produced by any codon arrangement was directly proportional to the degree of in vivo ribosome stalling at synonymous codons. Synonymous codon suppressors that corrected the effect of a translation-defective synonymous flgM allele were restricted to two codons flanking the translation-defective codon. The various codon arrangements had no apparent effects on flgM mRNA stability or predicted mRNA secondary structures. Our data suggest that efficient mRNA translation is determined by a triplet-of-triplet genetic code. That is, the efficiency of translating a particular codon is influenced by the nature of the immediately adjacent flanking codons. A model explains these codon-context effects by suggesting that codon recognition by elongation factor-bound aminoacyl-tRNA is initiated by hydrogen bond interactions between the first two nucleotides of the codon and anticodon and then is stabilized by base-stacking energy over three successive codons.

Type-III secretion pore formed by flagellar protein FliP.

During assembly of the bacterial flagellum, protein subunits that form the exterior structures are exported through a specialized secretion apparatus energized by the proton gradient. This category of protein transport, together with the similar process that occurs in the injectisomes of gram-negative pathogens, is termed type-III secretion. The membrane-embedded part of the flagellar export apparatus contains five essential proteins: FlhA, FlhB, FliP, FliQ and FliR. Here, we have undertaken a variety of experiments that together support the proposal that the protein-conducting conduit is formed primarily, and possibly entirely, by FliP. Chemical modification experiments demonstrate that positions near the center of certain FliP trans-membrane (TM) segments are accessible to polar reagents. FliP expression sensitizes cells to a number of chemical agents, and mutations at predicted channel-facing positions modulate this effect. Multiple assays are used to show that FliP suffices to form a channel that can conduct a variety of medium-sized, polar molecules. Conductance properties are strongly modulated by mutations in a methionine-rich loop that is predicted to lie at the inner mouth of the channel, which might form a gasket around cargo molecules undergoing export. The results are discussed in the framework of an hypothesis for the architecture and action of the cargo-conducting part of the type-III secretion apparatus.

Mechanism of type-III protein secretion: Regulation of FlhA conformation by a functionally critical charged-residue cluster.

The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram-negative pathogens to export virulence factors into host cells. This mode of protein export is termed type-III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identify residues R147, R154 and D158 of FlhA as most critical. These lie in a small, well-conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5. Two-hybrid experiments demonstrate self-interaction of the domain, and targeted cross-linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA.

Thailandamide, a Fatty Acid Synthesis Antibiotic That Is Coexpressed with a Resistant Target Gene.

Microbes encode many uncharacterized gene clusters that may produce antibiotics and other bioactive small molecules. Methods for activating these genes are needed to explore their biosynthetic potential. A transposon containing an inducible promoter was randomly inserted into the genome of the soil bacterium Burkholderia thailandensis to induce antibiotic expression. This screen identified the polyketide/nonribosomal peptide thailandamide as an antibiotic and discovered its regulator, AtsR. Mutants of Salmonella resistant to thailandamide had mutations in the accA gene for acetyl coenzyme A (acetyl-CoA) carboxylase, which is one of the first enzymes in the fatty acid synthesis pathway. A second copy of accA in the thailandamide synthesis gene cluster keeps B. thailandensis resistant to its own antibiotic. These genetic techniques will likely be powerful tools for discovering other unusual antibiotics.

"Lost in translation: Seeing the forest by focusing on the trees".

A complex process translates messenger RNA (mRNA) base sequence into protein amino acid sequence. Transfer RNAs must recognize 3-base codons in the mRNA to insert the correct amino acids into the growing protein. Codon degeneracy makes decoding complicated in that multiple (synonymous) triplets can encode a single amino acid and multiple tRNAs can have the same anticodon. Over the last twenty years, new developments in structural biology, genome sequencing and bioinformatics has elucidated the intricacies of the ribosome structure and the details of the translation process. High throughput analyses of sequence information support the idea that mRNA folding has a major effect on expression for codons at the 5'-end of mRNA (N-terminal region of a polypeptide). Despite a forest of sequence data, significant details of the complex translation process can escape detection. However, a sensitive translation assay has allowed a single tree in this forest to be revealing.

Molecular ruler determines needle length for the Salmonella Spi-1 injectisome.

The type-III secretion (T3S) systems of bacteria are part of self-assembling nanomachines: the bacterial flagellum that enables cells to propel themselves through liquid and across hydrated surfaces, and the injectisome that delivers pathogenic effector proteins into eukaryotic host cells. Although the flagellum and injectisome serve different purposes, they are evolutionarily related and share many structural similarities. Core features to these T3S systems are intrinsic length control mechanisms for external cellular projections: the hook of the flagellum and the injectisome needle. We present evidence that the Spi-1 injectisome, like the Salmonella flagellar hook, uses a secreted molecular ruler, InvJ, to determine needle length. This result supports a universal length control mechanism using molecular rulers for T3S systems.

High-resolution mapping defines the cooperative architecture of Polycomb response elements.

Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb response elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of nonhistone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor ADF1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished by enhanced Polycomb recruitment both to PREs and to target promoters of repressed genes. These results suggest that the stability of multifactor complexes at promoters and regulatory elements is a crucial aspect of developmentally regulated gene expression.

ATPase-independent type-III protein secretion in Salmonella enterica.

Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.

The effects of codon context on in vivo translation speed.

We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called "silent" codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5'- and 3'- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.

Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli.

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 "degenerate" enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.

The effect of cell growth phase on the regulatory cross-talk between flagellar and Spi1 virulence gene expression.

The flagellar regulon controls Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD4C2 transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1(flhDC) and P5(flhDC), were functional in a wild-type background, while P6(flhDC) was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1(flhDC) initiates flagellar assembly and a negative autoregulatory loop through FlhD4C2-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1(flhDC) also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD4C2 transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.

FliT selectively enhances proteolysis of FlhC subunit in FlhD4C2 complex by an ATP-dependent protease, ClpXP.

We previously reported that the ClpXP ATP-dependent protease specifically recognizes and degrades the flagellar master transcriptional activator complex, FlhD4C2, to negatively control flagellar biogenesis. The flagellum-related protein, FliT, is also a negative regulator of flagellar regulon by inhibiting the binding of FlhD4C2 to the promoter DNA. We have found a novel pathway of FliT inhibition of FlhD4C2 activity connected to ClpXP proteolysis. An in vitro degradation assay using purified proteins shows that FliT selectively increases ClpXP proteolysis of the FlhC subunit in the FlhD4C2 complex. FliT behaves specifically to ClpXP-dependent proteolysis of FlhC. An in vitro interaction assay detects the ternary complex of FliT-FlhD4C2-ClpX. FliT promotes the affinity of ClpX against FlhD4C2 complex, whereas FliT does not directly interact with ClpX. Thus, FliT interacts with the FlhC in FlhD4C2 complex and increases the presentation of the FlhC recognition region to ClpX. The DNA-bound form of FlhD4C2 complex is resistant to ClpXP proteolysis. We suggest that the role of FliT in negatively controlling the flagellar gene expression involves increasing free molecules of FlhD4C2 sensitive to ClpXP proteolysis by inhibiting the binding to the promoter DNA as well as enhancing the selective proteolysis of FlhC subunit by ClpXP.

Differential transmembrane domain GXXXG motif pairing impacts major histocompatibility complex (MHC) class II structure.

Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αß heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2(+) class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2(+) I-A(k) conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2(+) versus Ia.2(-) I-A(k) class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.