Anuradha Janakiraman

SitABCD Is the Alkaline Mn2+ Transporter of Salmonella enterica Serovar Typhimurium

MntH, a bacterial homolog of the mammalian natural resistance-associated macrophage protein 1 (Nramp1), is a primary Mn2+ transporter of Salmonella enterica serovar Typhimurium and Escherichia coli. S. enterica serovar Typhimurium MntH expression is important for full virulence; however, strains carrying an mntH deletion are only partially attenuated and display no obvious signs of Mn2+ deficiency. We noted that promoter sequences for mntH and for the putative Fe2+ transporter sitABCD appeared to have the same regulatory element responsive to Mn2+ and so hypothesized that sitABCD could transport Mn2+ with high affinity. We have now characterized transport by SitABCD in S. enterica serovar Typhimurium using 54Mn2+ and 55Fe2+ and compared its properties to those of MntH. SitABCD mediates the influx of Mn2+ with an apparent affinity (Ka) identical to that of MntH, 0.1 μM. It also transports Fe2+ but with a Ka 30 to 100 times lower, 3 to 10 μM. Inhibition of 54Mn2+ transport by Fe2+ and of 55Fe2+ transport by Mn2+ gave inhibition constants comparable to each cations Ka for influx. Since micromolar concentrations of free Fe2+ are improbable in a biological system, we conclude that SitABCD functions physiologically as a Mn2+ transporter. The cation inhibition profiles of SitABCD and MntH are surprisingly similar for two structurally and energetically unrelated transporters, with a Cd2+ Ki of ≈1 μM and a Co2+ Ki of ≈20 μM and with Ni2+, Cu2+, and Fe3+ inhibiting both transporters only at concentrations of >0.1 mM. The one difference is that Zn2+ exhibits potent inhibition of SitABCD (Ki = 1 to 3 μM) but inhibits MntH weakly (Ki > 50 μM). We have previously shown that MntH transports Mn2+ most effectively under acidic conditions. In sharp contrast, SitABCD has almost no transport capacity at acid pHs and optimally transports Mn2+ at slightly alkaline pHs. Overall, coupled with evidence that each transporter is multiply but distinctly regulated at the transcriptional level, the distinct transport properties of MntH versus SitABCD suggest that each transporter may be specialized for Mn2+ uptake in different physiological environments.

IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed

Asymmetric localization of proteins is essential to many biological functions of bacteria. Shigella IcsA, an outer membrane protein, is localized to the old pole of the bacillus, where it mediates assembly of a polarized actin tail during infection of mammalian cells. Actin tail assembly provides the propulsive force for intracellular movement and intercellular dissemination. Localization of IcsA to the pole is independent of the amino‐terminal signal peptide (Charles, M., Perez, M., Kobil, J.H., and Goldberg, M.B., 2001, Proc Natl Acad Sci USA 98: 9871–9876) suggesting that IcsA targeting occurs in the bacterial cytoplasm and that its secretion across the cytoplasmic membrane occurs only at the pole. Here, we characterize the mechanism by which IcsA is secreted across the cytoplasmic membrane. We present evidence that IcsA requires the SecA ATPase and the SecYEG membrane channel (translocon) for secretion. Our data suggest that YidC is not required for IcsA secretion. Furthermore, we show that polar localization of IcsA is independent of SecA. Finally, we demonstrate that while IcsA requires the SecYEG translocon for secretion, components of this apparatus are uniformly distributed within the membrane. Based on these data, we propose a model for coordinate polar targeting and secretion of IcsA at the bacterial pole.

Identification of ZapD as a Cell Division Factor That Promotes the Assembly of FtsZ in Escherichia coli

The tubulin homolog FtsZ forms a polymeric membrane-associated ring structure (Z ring) at midcell that establishes the site of division and provides an essential framework for the localization of a multiprotein molecular machine that promotes division in Escherichia coli. A number of regulatory proteins interact with FtsZ and modulate FtsZ assembly/disassembly processes, ensuring the spatiotemporal integrity of cytokinesis. The Z-associated proteins (ZapA, ZapB, and ZapC) belong to a group of FtsZ-regulatory proteins that exhibit functionally redundant roles in stabilizing FtsZ-ring assembly by binding and bundling polymeric FtsZ at midcell. In this study, we report the identification of ZapD (YacF) as a member of the E. coli midcell division machinery. Genetics and cell biological evidence indicate that ZapD requires FtsZ but not other downstream division proteins for localizing to midcell, where it promotes FtsZ-ring assembly via molecular mechanisms that overlap with ZapA. Biochemical evidence indicates that ZapD directly interacts with FtsZ and promotes bundling of FtsZ protofilaments. Similarly to ZapA, ZapB, and ZapC, ZapD is dispensable for division and therefore belongs to the growing group of FtsZ-associated proteins in E. coli that aid in the overall fitness of the division process.

Identification and characterization of ZapC: a stabilizer of the FtsZ-ring in Escherichia coli

In Escherichia coli, spatiotemporal control of cell division occurs at the level of the assembly/disassembly process of the essential cytoskeletal protein FtsZ. A number of regulators interact with FtsZ and modulate the dynamics of the assembled FtsZ ring at the midcell division site. In this article, we report the identification of an FtsZ stabilizer, ZapC (Z-associated protein C), in a protein localization screen conducted with E. coli. ZapC colocalizes with FtsZ at midcell and interacts directly with FtsZ, as determined by a protein-protein interaction assay in yeast. Cells lacking or overexpressing ZapC are slightly elongated and have aberrant FtsZ ring morphologies indicative of a role for ZapC in FtsZ regulation. We also demonstrate the ability of purified ZapC to promote lateral bundling of FtsZ in a sedimentation reaction visualized by transmission electron microscopy. While ZapC lacks sequence similarity with other nonessential FtsZ regulators, ZapA and ZapB, all three Zap proteins appear to play an important role in FtsZ regulation during rapid growth. Taken together, our results suggest a key role for lateral bundling of the midcell FtsZ polymers in maintaining FtsZ ring stability during division.

FtsZ Ring Stability: of Bundles, Tubules, Crosslinks, and Curves

The first step in bacterial cytokinesis is the assembly of a stable but dynamic cytokinetic ring made up of the essential tubulin homolog FtsZ at the future site of division. Although FtsZ and its role in cytokinesis have been studied extensively, the precise architecture of the in vivo medial FtsZ ring (Z ring) is not well understood. Recent advances in superresolution imaging suggest that the Z ring comprises short, discontinuous, and loosely bundled FtsZ polymers, some of which are tethered to the membrane. A diverse array of regulatory proteins modulate the assembly, stability, and disassembly of the Z ring via direct interactions with FtsZ. Negative regulators of FtsZ play a critical role in ensuring the accurate positioning of FtsZ at the future site of division and in maintaining Z ring dynamics by controlling FtsZ polymer assembly/disassembly processes. Positive regulators of FtsZ are essential for tethering FtsZ polymers to the membrane and promoting the formation of stabilizing lateral interactions, permitting assembly of a mature Z ring. The past decade has seen the identification of several factors that promote FtsZ assembly, presumably through a variety of distinct molecular mechanisms. While a few of these proteins are broadly conserved, many positive regulators of FtsZ assembly are limited to small groups of closely related organisms, suggesting that FtsZ assembly is differentially modulated across bacterial species. In this review, we focus on the roles of positive regulators in Z ring assembly and in maintaining the integrity of the cytokinetic ring during the early stages of division.

Transcriptional Regulation of sitABCD of Salmonella enterica Serovar Typhimurium by MntR and Fur

Salmonella enterica serovar Typhimurium has two manganese transport systems, MntH and SitABCD. MntH is a bacterial homolog of the eukaryotic natural resistance-associated macrophage protein 1 (Nramp1), and SitABCD is an ABC-type transporter. Previously we showed that mntH is negatively controlled at the transcriptional level by the trans-acting regulatory factors, MntR and Fur. In this study, we examined the transcriptional regulation of sitABCD and compared it to the transcriptional regulation of mntH by constructing lacZ fusions to the promoter regions with and without mutations in putative MntR and/or Fur binding sites. The presence of Mn caused transcriptional repression of the sitABCD and mntH promoters primarily via MntR, but Fur was also capable of some repression in response to Mn. Likewise, Fe in the medium repressed transcription of both sit and mntH primarily via Fur, although MntR was also involved in this response. Transcriptional control by MntR and Fur was disrupted by site-specific mutations in the putative MntR and Fur binding sites, respectively. Transcription of the sit operon was also affected by the oxygen level and growth phase, but the increased expression observed under high oxygen conditions and higher cell densities is consistent with decreased availability of metals required for repression by the metalloregulatory proteins.

A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters in Shigella

Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of E. coli. DnaK was also required for IcsA secretion at the pole in Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.

A novel paradigm of fatty acid beta-oxidation exemplified by the thioesterase-dependent partial degradation of conjugated linoleic acid that fully supports growth of Escherichia coli.

An alternative pathway of beta-oxidation for unsaturated fatty acids was studied in Escherichia coli. 9- cis,11- trans-Octadecadienoic acid (conjugated linoleic acid), a potential substrate of this pathway, was shown to support growth of E. coli in the absence of any other carbon source. The identification of 3,5-dodecadienoic acid in the growth medium revealed the partial beta-oxidation of conjugated linoleic acid to 3,5-dodecadienoyl-CoA, which was hydrolyzed to 3,5-dodecadienoic acid and released from cells. The involvement of acyl-CoA thioesterases in this process was evaluated by determining the substrate specificity of thioesterase II and comparing it with that of a novel thioesterase (thioesterase III) and by assessing mutant strains devoid of one or both of these thioesterases for growth on conjugated linoleic acid. Both thioesterases were highly active with 3,5-dodecadienoyl-CoA as substrate. A deficiency of either thioesterase decreased the growth rate of cells on conjugated linoleic acid but not on palmitic acid. The absence of both thioesterases reduced the cellular growth in a cumulative manner but did not abolish it. It is concluded that thioesterases II and III and at least one other thioesterase function in the partial degradation of conjugated linoleic acid via the thioesterase-dependent pathway of beta-oxidation, which provides all energy and carbon precursors required for the growth of E. coli.

Genetic Reporter System for Positioning of Proteins at the Bacterial Pole

ABSTRACT Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells. IMPORTANCE Many proteins localize to specific sites within bacterial cells, and localization to these sites is frequently critical to proper protein function. The mechanisms that underlie protein localization are incompletely understood, in part because of the paucity of methods that allow saturation screening for mutants in which protein localization is altered. We developed a genetic reporter assay that enables screening of bacterial populations for changes in localization of proteins to the bacterial pole, and we demonstrate the utility of the system in identifying factors required for proper localization of the polar Shigella autotransporter protein IcsA. Using this method, we identify the conserved cell division protein FtsQ as being required for positioning of IcsA to the bacterial pole. We demonstrate further that the requirement for FtsQ for polar positioning applies to other autotransporters and that the method can be applied to polar proteins other than autotransporters. Many proteins localize to specific sites within bacterial cells, and localization to these sites is frequently critical to proper protein function. The mechanisms that underlie protein localization are incompletely understood, in part because of the paucity of methods that allow saturation screening for mutants in which protein localization is altered. We developed a genetic reporter assay that enables screening of bacterial populations for changes in localization of proteins to the bacterial pole, and we demonstrate the utility of the system in identifying factors required for proper localization of the polar Shigella autotransporter protein IcsA. Using this method, we identify the conserved cell division protein FtsQ as being required for positioning of IcsA to the bacterial pole. We demonstrate further that the requirement for FtsQ for polar positioning applies to other autotransporters and that the method can be applied to polar proteins other than autotransporters.

Structural and Functional Analyses Reveal Insights into the Molecular Properties of the Escherichia coli Z Ring Stabilizing Protein, ZapC.

In Escherichia coli cell division is driven by the tubulin-like GTPase, FtsZ, which forms the cytokinetic Z-ring. The Z-ring serves as a dynamic platform for the assembly of the multiprotein divisome, which catalyzes membrane cleavage to create equal daughter cells. Several proteins effect FtsZ assembly, thereby providing spatiotemporal control over cell division. One important class of FtsZ interacting/regulatory proteins is the Z-ring-associated proteins, Zaps, which typically modulate Z-ring formation by increasing lateral interactions between FtsZ protofilaments. Strikingly, these Zap proteins show no discernable sequence similarity, suggesting that they likely harbor distinct structures and mechanisms. The 19.8-kDa ZapC in particular shows no homology to any known protein. To gain insight into ZapC function, we determined its structure to 2.15 Å and performed genetic and biochemical studies. ZapC is a monomer composed of two domains, an N-terminal α/β region and a C-terminal twisted β barrel-like domain. The structure contains two pockets, one on each domain. The N-domain pocket is lined with residues previously implicated to be important for ZapC function as an FtsZ bundler. The adjacent C-domain pocket contains a hydrophobic center surrounded by conserved basic residues. Mutagenesis analyses indicate that this pocket is critical for FtsZ binding. An extensive FtsZ binding surface is consistent with the fact that, unlike many FtsZ regulators, ZapC binds the large FtsZ globular core rather than C-terminal tail, and the presence of two adjacent pockets suggests possible mechanisms for ZapC-mediated FtsZ bundling.