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bioRxiv2017

Efficient hydrogen-dependent carbon dioxide reduction by Escherichia coli

The formate hydrogenlyase (FHL) complex of Escherichia coli is normally produced under anaerobic fermentative conditions when its physiological role is to oxidise formate to carbon dioxide and couple that reaction directly to the reduction of protons to molecular hydrogen. This forward reaction is the major route of hydrogen production by E. coli and the physiology, genetics and biochemistry of this process are reasonably well understood. However, studies of the reverse reaction - hydrogen-dependent carbon dioxide reduction - have been rather less extensive. Harnessing the alternative reverse reaction has the potential to unlock FHL as a carbon dioxide cycling enzyme, or could potentially lead to the development of bio-based carbon capture technologies. In this work, it is established that FHL can operate as a highly efficient CO2 reductase. A controllable pressure system was designed with the intention of maximising substrate availability to the FHL enzyme. By placing gaseous CO2 and H2 are under pressure (up to 10 bar), and by using a compartmentalised intact whole cell approach where the produced formate is excreted, the optimised experimental system was observed to convert 100 % of gaseous CO2 to formic acid and generate >500 mM formate in solution at an initial rate of 1.2 g formate per litre per hour.

microbiology↗

bioRxiv2017

The nucleoid as a scaffold for the assembly of bacterial signaling complexes

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.\n\nAUTHOR SUMMARYIn this work, we show that the cytoplasmic chemoreceptor of the Frz chemosensory system, FrzCD, does not bind the cytoplasmic membrane like most MCPs but bind the bacterial nucleoid directly, thus forming distributed protein clusters also containing the Frz kinase. In vitro and in vivo experiments show that DNA-binding is not sequence-specific and is mediated by a basic aminoacid sequence of the FrzCD N-terminal domain. The deletion of this motif abolishes FrzCD DNA-binding and cooperativity in the response to signals. This work shows the importance of the nucleoid in the organization and functioning of cytoplasmic signaling systems in bacteria.

microbiology↗

bioRxiv2017

The global biogeography of amino acid variants within a single SAR11 population is governed by natural selection

The diversity and geographical distribution of populations within major marine microbial lineages are largely governed by temperature and its co-variables. However, neither the mechanisms by which genomic heterogeneity emerges within a single population nor how it drives the partitioning of ecological niches are well understood. Here we took advantage of billions of metagenomic reads to study one of the most abundant and widespread microbial populations in the surface ocean. We characterized its substantial amount of genomic heterogeneity using single-amino acid variants (SAAVs), and identified systematic purifying selection and adaptive mechanisms governing non-synonymous variation within this population. Our Deep Learning analysis of SAAVs across metagenomes revealed two main ecological niches that reflect large-scale oceanic current temperatures, as well as six proteotypes demarcating finer-resolved niches. We identified significantly more protein variants in cold currents and an increased number of protein sweeps in warm currents, exposing a global pattern of alternating genomic diversity for this SAR11 population as it drifts along with surface ocean currents. Overall, the geographic partitioning of SAAVs suggests natural selection, rather than neutral evolution, is the main driver of the evolution of SAR11 in surface oceans.

microbiology↗

bioRxiv2017

Inhibition of NHEJ repair by type II-A CRISPR-Cas systems

CRISPR-Cas systems introduce double strand breaks into DNA of invading genetic material and use DNA fragments to acquire novel spacers during adaptation. Double strand breaks are the substrate of several bacterial DNA repair pathways, paving the way for interactions between them and CRISPR-Cas systems. Here, we hypothesized that non-homologous end joining (NHEJ) interferes with type II CRISPR-Cas systems. We tested this idea by studying the patterns of co-occurrence of the two systems in bacterial genomes. We found that NHEJ and type II-A CRISPR-Cas systems only co-occur once among 5563 fully sequenced prokaryotic genomes. We investigated experimentally the possible molecular interactions causing this negative association using the NHEJ pathway from Bacillus subtilis and the type II-A CRISPR-Cas systems from Streptococcus thermophilus and Streptococcus pyogenes. Our results suggest that the NHEJ system has no effect on type II-A CRISPR-Cas interference and adaptation. On the other hand, we provide evidence for the inhibition of NHEJ repair by the Csn2 protein from type II-A CRISPR-Cas system. Our findings give insights on the complex interactions between CRISPR- Cas systems and repair mechanisms in bacteria and contribute to explain the scattered distribution of CRISPR-Cas systems in bacterial genomes.

microbiology↗

bioRxiv2017

The Epstein-Barr Virus miR-BHRF1 microRNAs Regulate Viral Gene Expression in cis

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant ({triangleup}123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of primiR-BHRF1-2 and 1-3 in the {triangleup}123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the {triangleup}123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis.\n\nHighlightsO_LIThe EBV miR-BHRF1 microRNAs do not up upregulate B cell growth in trans.\nC_LIO_LIEBNA-LP expression is downregulated by pri-miR-BHRF1-2 and 1-3 acting in cis.\nC_LIO_LILoss of miR-BHRF1-2 and 1-3 causes EBNA-LP overexpression and inhibits B cell growth.\nC_LIO_LINovel alternative splicing of EBV Cp/Wp transcripts was identified.\nC_LI

microbiology↗

bioRxiv2017

Human placental syncytiotrophoblasts restrict Toxoplasma gondii vertical transmission at two distinct stages and induce CCL22 in response to infection

Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human mid-gestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at distinct stages of the parasite lytic cycle--at the time of attachment and also during intracellular replication. Utilizing comparative RNAseq transcriptional profiling, we also show that human placental trophoblasts at both mid- and late-stages of gestation induce the chemokine CCL22 in response to T. gondii infection, which relies on the secretion of parasite effector(s). Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy, and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment.\n\nSignificance statementToxoplasma gondii is a major source of congenital disease worldwide and must breach the placental barrier to be transmitted from maternal blood to the developing fetus. The events associated with the vertical transmission of T. gondii are largely unknown. Here, we show that primary human syncytiotrophoblasts, the fetal-derived cells that comprise the primary placental barrier, restrict T. gondii infection at two distinct stages of the parasite life cycle and respond to infection through the induction of the chemokine CCL22. Collectively, our findings provide important insights into the mechanisms by which human syncytiotrophoblasts restrict T. gondii infection at early and late stages of human pregnancy and identify the placental-enriched signaling pathways induced in response to infection.

microbiology↗

bioRxiv2017

The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructure in vitro

The bacterial tubulin FtsZ2 polymerizes to form a discontinuous cytokinetic ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL) and a C-terminal conserved -helix (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The dynamic turnover and treadmilling of clusters of FtsZ protofilaments guides cell wall synthesis and constriction. The nature and regulation of the interactions that result in the assembly of protofilaments into dynamic clusters is unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro. FtsZ lacking its CTL ({Delta}CTL) shows dramatically increased propensity to form long multifilament bundles compared to wildtype (WT). {Delta}CTL has reduced GTP hydrolysis rate compared to WT. However, reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts {Delta}CTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between Z-ring structure and function in bacterial cytokinesis.

microbiology↗

bioRxiv2017

An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e., \"orphan\") operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen P. aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets.

microbiology↗

bioRxiv2017

Detection of live mycobacteria with a solvatochromic trehalose probe for point-of-care tuberculosis diagnosis

AbstractTuberculosis (TB) is the leading cause of death from an infectious bacterial disease. Poor diagnostic tools to detect active disease plague TB control programs and affect patient care. Accurate detection of live Mycobacterium tuberculosis (Mtb), the causative agent of TB, will improve TB diagnosis and patient treatment. We report that live mycobacteria can be specifically detected with a fluorogenic trehalose analog. We designed a 4-N,N-dimethylamino-1,8- naphthalimide-trehalose (DMN-Tre) conjugate that undergoes >700-fold fluorescence increase when transitioned from aqueous to hydrophobic environments. This enhancement occurs upon metabolic conversion of DMN-Tre to trehalose monomycolate and incorporation into the outer membrane. DMN-Tre labeling enabled the rapid, no-wash visualization of mycobacterial and corynebacterial species without nonspecific labeling of Gram-positive or -negative bacteria. DMN-Tre labeling was selective for live mycobacteria and was reduced by treatment with TB drugs. Lastly, DMN-Tre labeled Mtb in TB-positive sputum samples suggesting this operationally simple method may be deployable for TB diagnosis.

microbiology↗

bioRxiv2017

The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135

5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be microbially degraded. However, the physiological, biochemical, and genetic foundation of the microbial catabolism of 5HPA remains unknown. In this study, a gene cluster hpa (which is involved in degradation of 5HPA in Alcaligenes faecalis JQ135) was cloned and HpaM was identified as a novel monocomponent FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) of Pseudomonas putida KT2440. hpaM was heterologously expressed in E. coli BL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography. HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the presence of FAD and NADH, but not of FMN and NADPH. The apparent Km values of HpaM toward 5HPA and NADH were 45.4 and 37.8 , respectively. Results of gene deletion and complementation showed that hpaM was essential for 5HPA degradation in Alcaligenes faecalis JQ135.\n\nImportancePyridine derivatives are ubiquitous in nature and important chemical materials that are currently widely used in agriculture, pharmaceutical, and chemical industries. Thus, the microbial degradation and transformation mechanisms of pyridine derivatives received considerable attention. Decarboxylative hydroxylation was an important degradation process in pyridine derivatives, and previously reported decarboxylative hydroxylations happened in the C3 of the pyridine ring. In this study, we cloned the gene cluster hpa, which is responsible for 5HPA degradation in Alcaligenes faecalis JQ135, thus identifying a novel monocomponent FAD-dependent monooxygenase HpaM. Unlike 3-decarboxylative monooxygenases, HpaM catalyzed decarboxylative hydroxylation in the C2 of the pyridine ring in 5-hydroxypicolinic acid. These findings deepen our understanding of the molecular mechanism of microbial degradation of pyridine derivatives. Furthermore, HpaM offers potential for applications to transform useful pyridine derivatives.

microbiology↗

bioRxiv2017

A dynamic microbial community with high functional redundancy inhabits the cold, oxic subseafloor aquifer

The rock-hosted subseafloor crustal aquifer harbors a reservoir of microbial life that may influence global marine biogeochemical cycles. Here we utilized genomic reconstruction of crustal fluid samples from North Pond, located on the flanks of the Mid-Atlantic Ridge, a site with cold, oxic subseafloor fluid circulation within the upper basement. Twenty-one samples were collected during a two-year period at three different depths and two locations with the basaltic aquifer to examine potential microbial metabolism and community dynamics. We observed minor changes in the geochemical signatures over the two years, yet a dynamic microbial community was present in the crustal fluids that underwent large shifts in the dominant taxonomic groups. An analysis of 195 metagenome-assembled genomes (MAGs) were generated from the dataset and revealed a connection between litho- and autotrophic processes, linking carbon fixation to the oxidation of sulfide, sulfur, thiosulfate, hydrogen, and ferrous iron in a diverse group of microorganisms. Despite oxic conditions, analysis of the MAGs indicated that members of the microbial community were poised to exploit hypoxic or anoxic conditions through the use of microaerobic cytochromes and alternative electron acceptors. Temporal and spatial trends from the MAGs revealed a high degree of functional redundancy that did not correlate with the shifting microbial community membership, suggesting functional stability in mediating subseafloor biogeochemical cycles.

microbiology↗

bioRxiv2017

First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (formerly Scedosporium prolificans)

Here we describe the sequencing and assembly of the pathogenic fungus Lomentospora prolificans using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads. The resulting assembly is highly contiguous, containing a total of 37,630,066 bp with over 98% of the sequence in just 26 scaffolds. Annotation identified 8,656 protein-coding genes. Pulsed-field gel analysis suggests that this organism contains at least 7 and possibly 11 chromosomes, the two longest of which have sizes corresponding closely to the sizes of the longest scaffolds, at 6.6 and 5.7 Mb.

microbiology↗

bioRxiv2017

Anti-infective potential of a quorum modulatory polyherbal extract (Panchvalkal) against certain pathogenic bacteria

Anti-infective potential of a polyherbal ayurvedic formulation namely panchvalkal was assayed against three pathogenic bacteria. This formulation was found to exert quorum-modulatory effect on Chromobacterium violaceum, Serratia marcescens, and Staphylococcus aureus at 250-750 g/mL. Besides altering production of the quorum sensing-regulated pigments in these bacteria, the test formulation also had in vitro effect on antibiotic susceptibility, catalase activity and hemolytic potential of the pathogens. In vivo assay confirmed the protective effect of this panchvalkal formulation on Caenorhabditis elegans, when challenged with the pathogenic bacteria. Repeated exposure of S. aureus to panchvalkal did not induce resistance in this bacterium. To the best of our awareness, this the first report on quorum-modulatory potential of panchvalkal formulation, validating the anti-infective potential and moderate prebiotic property of this polyherbal preparation.

microbiology↗

bioRxiv2017

dRNA-seq implicates sulfide as master regulator of S(0) metabolism in Chlorobaculum tepidum and other green sulfur bacteria

The green sulfur bacteria (Chlorobiaceae) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S(0), and thiosulfate) as electron donors for photoautotrophic growth. Chlorobaculum tepidum, the model system for the Chlorobiaceae, both produces and consumes extracellular S(0) globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to Cba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e. specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA-seq (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S(0), and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor dependent transcriptional regulation. These motifs were conserved across homologous Chlorobiaceae promoters. Two lines of evidence suggest that sulfide mediated repression is the dominant regulatory mode in Cba. tepidum. First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the gene CT1277, encoding a putative regulatory protein, leads to constitutive over-expression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in Cba. tepidum and the Chlorobiaceae. Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in Cba. tepidum and perhaps other Chlorobiaceae.\n\nImportanceElemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium Chlorobaculum tepidum both produces and consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of Chlorobaculum tepidum on elemental sulfur, and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This study identifies new genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this study paves the way for the development of synthetic biology in Chlorobaculum tepidum and other Chlorobiaceae by providing a comprehensive identification of promoter elements to control gene expression, a key element of strain engineering.

microbiology↗

bioRxiv2017

Multi-clonal evolution of MDR/XDR M. tuberculosis in a high prevalence setting in Papua New Guinea over three decades

An outbreak of multi-drug resistant tuberculosis has been reported on Daru Island, Papua New Guinea. The Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. We analyzed 100 isolates using whole genome sequencing and found 95 belonged to a single modern Beijing strain cluster. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with virulence potentially enhanced by a mycP1 mutation. The outbreak cluster demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2%) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multidrug resistance (MDR), observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance; with evidence of local transmission of extensively-drug resistant (XDR) strains from 2009. These findings underscore the importance of whole-genome sequencing in informing an effective public health response to MDR/XDR M. tuberculosis.

microbiology↗

bioRxiv2017

Geobacter sulfurreducens extracellular multiheme cytochrome PgcA facilitates respiration to Fe(III) oxides but not electrodes

Extracellular cytochromes are hypothesized to facilitate the final steps of electron transfer between the outer membrane of the metal-reducing bacterium Geobacter sulfurreducens and solid-phase electron acceptors such as metal oxides and electrode surfaces during the course of respiration. The triheme c-type cytochrome PgcA exists in the extracellular space of G. sulfurreducens, and is one of many multiheme c-type cytochromes known to be loosely bound to the bacterial outer surface. Deletion of pgcA using a markerless method resulted in mutants unable to transfer electrons to Fe(III) and Mn(IV) oxides; yet the same mutants maintained the ability to respire electrode surfaces and soluble Fe(III) citrate. When expressed and purified from Shewanella oneidensis, PgcA demonstrated a primarily alpha helical structure, three bound hemes, and was processed into a shorter 41 kDa form lacking the lipodomain. Purified PgcA bound Fe(III) oxides, but not magnetite, and when PgcA was added to cell suspensions of G. sulfurreducens, PgcA accelerated Fe(III) reduction similar to addition of FMN. Addition of soluble PgcA to {triangleup}pgcA mutants also restored Fe(III) reduction. This report highlights a distinction between proteins involved in extracellular electron transfer to metal oxides and poised electrodes, and suggests a specific role for PgcA in facilitating electron transfer at mineral surfaces.

microbiology↗

bioRxiv2017

Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome

Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA-ParB-parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ~8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ~10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ~500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

microbiology↗

bioRxiv2017

A genetic screen suggests an alternative mechanism for inhibition of SecA by azide

Sodium azide prevents bacterial growth by inhibiting the activity of SecA, which is required for translocation of proteins across the cytoplasmic membrane. Azide inhibits ATP turnover in vitro, but its mechanism of action in vivo is unclear. To investigate how azide inhibits SecA in cells, we used transposon directed insertion-site sequencing (TraDIS) to screen a library of transposon insertion mutants for mutations that affect the susceptibility of E. coli to azide. Insertions disrupting components of the Sec machinery generally increased susceptibility to azide, but insertions truncating the C-terminal tail (CTT) of SecA decreased susceptibility of E. coli to azide. Treatment of cells with azide caused increased aggregation of the CTT, suggesting that azide disrupts its structure. Analysis of the metal-ion content of the CTT indicated that SecA binds to iron and the azide disrupts the interaction of the CTT with iron. Azide also disrupted binding of SecA to membrane phospholipids, as did alanine substitutions in the metal-coordinating amino acids. Furthermore, treating purified phospholipid-bound SecA with azide in the absence of added nucleotide disrupted binding of SecA to phospholipids. Our results suggest that azide does not inhibit SecA by inhibiting the rate of ATP turnover in vivo. Rather, azide inhibits SecA by causing it to \"backtrack\" from the ADP-bound to the ATP-bound conformation, which disrupts the interaction of SecA with the cytoplasmic membrane.\n\nSignificance statementSecA is a bacterial ATPase that is required for the translocation of a subset of secreted proteins across the cytoplasmic membrane. Sodium azide is a well-known inhibitor of SecA, but its mechanism of action in vivo is poorly understood. To investigate this mechanism, we examined the effect of azide on the growth of a library of [~]1 million transposon insertion mutations. Our results suggest that azide causes SecA to backtrack in its ATPase cycle, which disrupts binding of SecA to the membrane and to its metal cofactor, which is iron. Our results provide insight into the molecular mechanism by which SecA drives protein translocation and how this essential biological process can be disrupted.

microbiology↗