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.

Estimating uninsured and underinsured women eligible for Minnesota's Breast Cancer Screening Program.

Since its inception in 1991, the mission of the National Breast and Cervical Cancer Early Detection Program's (NBCCEDP) mission is to improve access to mammography. This program has demonstrated evidence showing that it has improved breast cancer screening rates for women who are uninsured and underinsured. However, the literature has shown that NBCCEDP screenings are decreasing, and only reach a portion of eligible women. Reliable estimates at the sub-county level are needed to identify and reach eligible women. Our work builds upon previous estimates by integrating uninsured and insurance status into spatially adaptive filters. We use spatially adaptive filters to create small area estimates of standardized incidence ratios describing the utilization rate of NBCCEDP services in Minnesota. We integrate the American Community Survey (2010-2014) insurance status data to account for the percentage that an individual is uninsured. We test five models that integrate insurance status by age, sex, and race/ethnicity. Our composite model, which adjusts for age, sex, and race/ethnicity insurance statuses, reduces 95% of the estimation error. We estimate that there approximately 49,913.7 women eligible to receive services for Minnesota. We also create small geography (i.e., county and sub-county) estimates for Minnesota. The integration of the insurance data improved our utilization estimate. The development of these methods will allow state programs to more efficiently use their resources and understand their reach.

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.

Establishment, Persistence, and Reactivation of Latent HIV-1 Infection in Renal Epithelial Cells.

HIV-1 persistence in different cell types presents the main obstacle to an HIV-1 cure. We have previously shown that the renal epithelium is a site of HIV-1 infection and that the kidney represents a separate viral compartment from blood. Whether renal cells can harbor latent virus that can be reactivated upon treatment with latency reversing agents (LRAs) is unknown. To address this question, we developed an in vitro HIV-1 latency model in renal tubule epithelial (RTE) cells using a dual color HIV-1 reporter virus, R7/E-/GFP/EF1a-mCherry (R7GEmC), and evaluated the effect of LRAs, both as single agents and in combination, on viral reactivation. Our data show that HIV-1 can establish latency in RTE cells early postinfection. While the pool of latently infected cells expanded overtime, the percentage of productively infected cells declined. Following LRA treatment only a small fraction of latently infected cells, both T cells and RTE cells, could be reactivated, and the drug combinations more effective in reactivating HIV transcription in RTE cells differed from those more active in T cells. Our study demonstrates that HIV can establish latency in RTE cells and that current LRAs are only marginally effective in inducing HIV-1 reactivation. This suggests that further study of LRA dynamics in non-T cells may be warranted to assess the suitability of LRAs as a sterilizing cure strategy. IMPORTANCE Anti-retroviral therapy (ART) has dramatically reduced HIV-related morbidity and mortality. Despite this success, a number of challenges remain, including the long-term persistence of multiple, clinically latent viral reservoirs capable of reactivation in the absence of ART. As efforts proceed toward HIV eradication or functional cure, further understanding of the dynamics of HIV-1 replication, establishment of latency and mechanisms of reactivation in reservoirs harboring the virus throughout the body is necessary. HIV-1 can infect renal epithelial cells and the expression of viral genes in those cells contributes to the development of HIV associated nephropathy (HIVAN) in untreated individuals. The significance of our work is in developing the first model of HIV-1 latency in renal epithelial cells. This model enhances our understanding of HIV-1 latency and persistence in the kidney and can be used to screen candidate latency reversing agents.

Breast Cancer Champions: a peer-to-peer education and mobile mammography program improving breast cancer screening rates for women of African heritage.

INTRODUCTION: Nationally, women of African heritage die at higher rates from breast cancer than women of other races or ethnicities. We developed Breast Cancer Champions (BCC) a peer-to-peer education program, which recruited 12 women and deployed them into the community in August 2020 during the height of the COVID pandemic. BCC aims to improve breast cancer screening rates for women of African heritage through peer-to-peer education, which has proven successful for addressing cancer-related health disparities. METHODS: BCC community experts, or "Champions," are peer-to-peer educators that conduct awareness and screening events in their communities. Champion's education activities were tracked by bi-weekly check-in calls, which recorded the activity type, location, and the number of participants for each event. We used spatial and statistical analyses to determine the efficacy of the program at increasing screening rates for women within the area of Champion activity versus women outside of their activity area. RESULTS: Over 15 months, Champions conducted 245 in-person or online events to engage women in their community for screening. More women of African heritage were screened in areas Champions were active during the intervention compared to historical data comparing areas outside of the Champion activity in the prior 15 months (X 2 = 3.0845, p = 0.079). CONCLUSION: BCC successes could be attributed to pivoting to online community building when in-person events were restricted and enabling Champions to design and conduct their own events, which increased outreach possibilities. We demonstrate improved screening outcomes associated with an updated peer-to-peer education program.

Diagnosis or prognosis? An umbrella review of mid-trimester cervical length and spontaneous preterm birth.

BACKGROUND: Cervical length is widely used to assess a woman's risk of spontaneous preterm birth (SPTB). OBJECTIVES: To summarise and critically appraise the evidence from systematic reviews on the prognostic capacity of transvaginal sonographic cervical length in the second trimester in asymptomatic women with singleton or twin pregnancy. SEARCH STRATEGY: Searches were performed in Medline, Embase, CINAHL and grey literature from 1 January 1995 to 6 July 2021, including keywords 'cervical length', 'preterm birth', 'obstetric labour, premature', 'review' and others, without language restriction. SELECTION CRITERIA: We included systematic reviews including women who did not receive treatments to reduce SPTB risk. DATA COLLECTION AND ANALYSIS: From 2472 articles, 14 systematic reviews were included. Summary statistics were independently extracted by two reviewers, tabulated and analysed descriptively. The ROBIS tool was used to evaluate risk of bias of included systematic reviews. MAIN RESULTS: Twelve reviews performed meta-analyses: two were reported as systematic reviews of prognostic factor studies, ten used diagnostic test accuracy methodology. Ten systematic reviews were at high or unclear risk of bias. Meta-analyses reported up to 80 combinations of cervical length, gestational age at measurement and definition of preterm birth. Cervical length was consistently associated with SPTB, with a likelihood ratio for a positive test of 1.70-142. CONCLUSIONS: The ability of cervical length to predict SPTB is a prognostic research question; systematic reviews typically analysed diagnostic test accuracy. Individual participant data meta-analysis using prognostic factor research methods is recommended to better quantify how well transvaginal ultrasonographic cervical length can predict SPTB.

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.

Diffuse Small B-Cell Lymphoma: A High-Grade Malignancy.

The most common subtype of lymphoma in the dog is diffuse large B-cell lymphoma (DLBCL). The remaining forms of B-cell lymphoma in dogs are categorized as small-to-intermediate in size and include marginal zone, follicular, mantle cell, and small-cell lymphocytic lymphoma. Marginal zone lymphoma and follicular lymphoma have readily identifiable unique histologic features while other forms of small B-cell lymphoma in the dog are poorly described by histopathology. Forty-seven cases of nodal small B-cell lymphoma identified by flow cytometry (small cell size based on forward scatter) with concurrent histopathology were reviewed. These cases fell into 3 histologic subtypes: marginal zone lymphoma, follicular lymphoma, and a diffuse form of small B-cell lymphoma with consistent features. As a descriptive term, we refer to the latter subtype as diffuse small B-cell lymphoma (DSBCL) until it can be further characterized by gene expression profiling and other molecular tools. Clinical presentation of DSBCL was compared to cases of histologically confirmed DLBCL and clinical follow-up was obtained for 22 of the 27 cases of DSBCL. This subset of diffuse small B-cell lymphoma had an overall median survival of 140 days. The expression of CD21, class II MHC and CD25 by flow cytometry did not differ between DSBCL and the other histologic subtypes of small cell B-cell lymphoma making histopathology the only current method of classification.

The contemporary value of dedicated preterm birth clinics for high-risk singleton pregnancies: 15-year outcomes from a leading maternal centre.

OBJECTIVES: Preterm birth clinics provide dedicated obstetric care to women at high risk of spontaneous preterm birth (SPTB). There remains a lack of conclusive evidence to support the overall utility of such clinics, attributable to a paucity and heterogeneity of primary data. This study audits Australia's largest and oldest dedicated preterm birth clinic with the aim to add primary data to the area and offer opportunities for similar clinics to align practice. METHODS: A retrospective audit of referrals to the Preterm Labour Clinic at the Royal Women's Hospital, Melbourne, Australia, between 2004 and 2018 was conducted. 1,405 singleton pregnancies met inclusion criteria. The clinic's key outcomes, demographics, predictive tests and interventions were analysed. The primary outcomes were SPTB before 37, 34 and 30 weeks' gestation. RESULTS: The overall incidence of SPTB in the clinic was 21.2% (n=294). Linear regression showed reductions in the adjusted rates of overall SPTB and pre-viable SPTB (delivery <24 weeks) from 2004 (108%; 8%) to 2018 (65%; 2% respectively). Neonatal morbidity and post-delivery intensive care admission concurrently declined (p=0.02; 0.006 respectively). Rates of short cervix (cervical length <25 mm) increased over time (2018: 30.9%) with greater uptake of vaginal progesterone for treatment. Fetal fibronectin, mid-trimester short cervix, and serum alkaline phosphatase were associated with SPTB on logistic regression. CONCLUSIONS: Dedicated preterm birth clinics can reduce rates of SPTB, particularly deliveries before 24 weeks' gestation, and improve short-term neonatal outcomes in pregnant women at risk of preterm birth.

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.

Cervical length surveillance for predicting spontaneous preterm birth in women with uterine anomalies: A cohort study.

INTRODUCTION: Uterine anomalies occur in an estimated 5% of women and have been shown to confer a higher risk of spontaneous preterm birth (SPTB). A sonographically short cervix (<25 mm) is a risk indicator for SPTB, although its predictive utility has been little studied in this specific high-risk population. We aimed to assess the pregnancy outcomes and predictive ability of short cervix in a cohort of women with uterine anomalies attending a high-risk antenatal clinic. MATERIAL AND METHODS: This historical cohort study assessed all pregnancies in women with congenital uterine anomalies referred to the Preterm labor Clinic at the Royal Women's Hospital, Melbourne, Australia, between 2004 and 2013. Logistic and linear regressions and receiver-operator curves were used to examine associations between cervical length and preterm birth. RESULTS: SPTB (<37 weeks' gestation) occurred in 23% of the 86 pregnancies (n = 20); rates by subgroup were: unicornuate uterus 60% (n = 3/5), uterus didelphys 40% (n = 6/15), bicornuate uterus 18% (n = 9/51), septate uterus 13% (n = 2/15). Preterm prelabor rupture of membranes occurred in 55% of spontaneous preterm births and was not independently associated with the presence of cervical cerclage or ureaplasma urealyticum. Short cervical length was associated with SPTB in women with septate uterus. Short cervix at 24 weeks (not at 16 or 20 weeks) was moderately predictive of SPTB < 34 weeks. CONCLUSIONS: Women with uterine anomalies are at increased risk of spontaneous preterm birth, particularly those with unicornuate uterus or uterus didelphys, but cervical surveillance did not identify these cases. Short cervix may be associated with SPTB in women with septate uterus. Preterm prelabor rupture of membranes occurred in 55% of SPTB. More research is required into etiology to help determine appropriate monitoring and treatment.

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.

Financial incentives and proactive calling for reducing barriers to tobacco treatment among socioeconomically disadvantaged women: A factorial randomized trial.

Improved strategies and scalable interventions to engage low-socioeconomic status (SES) smokers in tobacco treatment are needed. We tested an intervention designed to connect low-SES smokers to treatment services, implemented through Minnesota's National Breast and Cervical Cancer Early Detection Program (Sage) in 2017; the trial was designed to last 3 months (July through October). Participants were female smokers who were 250% below the federal poverty level (randomized N = 3723; analyzed N = 3365). Using a factorial design, participants were randomized to six intervention groups consisting of a proactive call (no call vs call) and/or a financial incentive offered for being connected to treatment services ($0 vs $10 vs $20). Simple randomization was conducted using Stata v.13. All individuals received direct mail. Participants and staff were blinded to allocation. The outcome was connection via phone to QUITPLAN Services®, Minnesota's population-based cessation services. Groups that received $10 or $20 incentives had higher odds of treatment engagement compared to the no incentive group [respectively, OR = 1.94; 95% CI (1.19-3.14); OR = 2.18; 95% CI (1.36-3.51)]. Individuals that received proactive calls had higher odds of treatment engagement compared to individuals not called [OR = 1.59; 95% CI (1.11-2.29)]. Economic evaluation revealed that the $10 incentive, no call group had the best cost-benefit ratio compared to the no incentive, no call group. Direct mail with moderate incentives or proactive calling can successfully encourage connections to population-based tobacco treatment services among low-SES smokers. The intervention could be disseminated to similar programs serving low-SES populations. This trial is registered at ClinicalTrials.gov (NCT03760107).

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.