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1.  MntR(Rv2788) a transcriptional regulator that controls manganese homeostasis in Mycobacterium tuberculosis 
Molecular microbiology  2015;98(6):1168-1183.
The pathogenic mycobacterium Mycobacterium tuberculosis encodes two members of the DtxR/MntR family of metalloregulators, IdeR and SirR. IdeR represses gene expression in response to ferrous iron, and we here demonstrate that SirR (Rv2788), although also annotated as an iron-dependent repressor, functions instead as a manganese dependent transcriptional repressor and is therefore renamed MntR. MntR regulates transporters that promote manganese import and genes that respond to metal ion deficiency such as the esx3 system. Repression of manganese import by MntR is essential for survival of M. tuberculosis under conditions of high manganese availability, but mntR is dispensable during infection. In contrast, manganese import by MntH and MntABCD was found to be indispensable for replication of M. tuberculosis in macrophages. These results suggest that manganese is limiting in the host and that interfering with import of this essential metal may be an effective strategy to attenuate M. tuberculosis.
PMCID: PMC5157835  PMID: 26337157
2.  Protein acetylation dynamics in response to carbon overflow in Escherichia coli 
Molecular microbiology  2015;98(5):847-863.
In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl-phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose, and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. Since glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl-phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl-phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism.
PMCID: PMC4715485  PMID: 26264774
acetylation; acetyl phosphate; metabolism; post-translational regulation; proteomics
3.  Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter 
Molecular microbiology  2015;98(5):878-894.
MalFGK2 is an ATP-binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose-binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide-binding subunits (MalK dimer). This MBP-stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance (EPR) spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose-bound MBP unto MalFGK2 is facilitated by nucleotides and stabilizes a semi-open MalK dimer. Maltose-bound MBP promotes the transition to the semi-open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi-open MalK2 conformation by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP.
PMCID: PMC4715599  PMID: 26268698
ABC transporter; ATPase; conformational change; electron paramagnetic resonance (EPR); sugar transport; membrane reconstitution
4.  MinC/MinD copolymers are not required for Min function 
Molecular microbiology  2015;98(5):895-909.
In Escherichia coli, precise placement of the cytokinetic Z ring at midcell requires the concerted action of the three Min proteins. MinD activates MinC, an inhibitor of FtsZ, at least in part, by recruiting it to the membrane and targeting it to the Z ring, while MinE stimulates the MinD ATPase inducing an oscillation that directs MinC/MinD activity away from midcell. Recently, MinC and MinD were shown to form copolymers of alternating dimers of MinC and MinD and it was suggested that these copolymers are the active form of MinC/MinD. Here, we use MinD mutants defective in binding MinC to generate heterodimers with wild type MinD that are unable to form MinC/MinD copolymers. Similarly, MinC mutants defective in binding to MinD were used to generate heterodimers with wild type MinC that are unable to form copolymers. Such heterodimers are active and in the case of MinC were shown to mediate spatial regulation of the Z ring demonstrating that MinC/MinD copolymer formation is not required. Our results are consistent with a model in which a membrane anchored MinC/MinD complex is targeted to the Z ring through the conserved carboxy tail of FtsZ leading to breakage of FtsZ filaments.
PMCID: PMC4715711  PMID: 26268537
Z ring; MinC; MinD; copolymer
5.  The mycobacterial iron dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression 
Molecular microbiology  2015;98(5):864-877.
Emerging evidence indicates that precise regulation of iron (Fe) metabolism and maintenance of Fe homeostasis in Mycobacterium tuberculosis (Mtb) are essential for its survival and proliferation in the host. IdeR is a central transcriptional regulator of Mtb genes involved in Fe metabolism. While it is well understood how IdeR functions as a repressor, how it induces transcription of a subset of its targets is still unclear. We investigated the molecular mechanism of IdeR-mediated positive regulation of bfrB, the gene encoding the major Fe-storage protein of Mtb. We found that bfrB induction by Fe required direct interaction of IdeR with a DNA sequence containing four tandem IdeR-binding boxes located upstream of the bfrB promoter. Results of in vivo and in vitro transcription assays identified a direct repressor of bfrB, the histone-like protein Lsr2. IdeR counteracted Lsr2-mediated repression in vitro, suggesting that IdeR induces bfrB transcription by antagonizing the repressor activity of Lsr2. Together these results elucidate the main mechanism of bfrB positive regulation by IdeR and identify Lsr2 as a new factor contributing to Fe homeostasis in mycobacteria.
PMCID: PMC4879814  PMID: 26268801
6.  Essentiality of threonylcarbamoyladenosine (t6A), a universal tRNA modification, in bacteria 
Molecular microbiology  2015;98(6):1199-1221.
Threonylcarbamoyladenosine (t6A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t6A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNAIle-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t6A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t6A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t6A in tRNAs. Thus, although t6A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.
PMCID: PMC4963248  PMID: 26337258
tRNA; maturation; translation; modified nucleosides; t6A
7.  A repeat sequence domain of the ring-exported protein-1 of Plasmodium falciparum controls export machinery architecture and virulence protein trafficking 
Molecular microbiology  2015;98(6):1101-1114.
The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer’s clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer’s cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer’s cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer’s cleft stacks. The stacked Maurer’s clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer’s cleft sculpting.
PMCID: PMC4987487  PMID: 26304012
Trafficking; Virulence protein; Maurer’s clefts REX1; PfEMP1; KAHRP
8.  Macrophage cell death and transcriptional response are actively triggered by the fungal virulence factor Cbp1 during H. capsulatum infection 
Molecular microbiology  2015;98(5):910-929.
Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth, but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis.
PMCID: PMC5002445  PMID: 26288377
9.  The TatC component of the twin‐arginine protein translocase functions as an obligate oligomer 
Molecular Microbiology  2015;98(1):111-129.
The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in E scherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.
PMCID: PMC5102672  PMID: 26112072
10.  The γ-tubulin complex in Trypanosoma brucei: molecular composition, subunit interdependence and requirement for axonemal central pair protein assembly 
Molecular microbiology  2015;98(4):667-680.
The γ-tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, GCP2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex.
PMCID: PMC4636443  PMID: 26224545
11.  tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum 
Molecular microbiology  2015;98(4):681-693.
Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane, and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine, and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Ala-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, while formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.
PMCID: PMC4639916  PMID: 26235234
phospholipids; multiple peptide resistance factor (MprF); Actinobacteria; diacylglycerol; phosphatidylglycerol; aminoacyl-tRNA synthetase; transfer RNA (tRNA); amino acid; lipids; membrane proteins
12.  PfeT, a P1B4-type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication 
Molecular microbiology  2015;98(4):787-803.
Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4-type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4-type ATPases previously implicated in bacterial pathogenesis.
PMCID: PMC4648274  PMID: 26261021
13.  ArsP: a methylarsenite efflux permease 
Molecular microbiology  2015;98(4):625-635.
Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III)>Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus ArsP is the first identified efflux system specific for trivalent organoarsenicals.
PMCID: PMC4681449  PMID: 26234817
Methylarsenite; herbicide resistance; roxarsone; antimicrobial growth promoter; efflux permease
14.  Phytohormone sensing in the biotrophic fungus Ustilago maydis – the dual role of the transcription factor Rss1 
Molecular Microbiology  2016;102(2):290-305.
The phenolic compound salicylic acid (SA) is a key signalling molecule regulating local and systemic plant defense responses, mainly against biotrophs. Many microbial organisms, including pathogens, share the ability to degrade SA. However, the mechanism by which they perceive SA is unknown. Here we show that Ustilago maydis, the causal agent of corn smut disease, employs a so far uncharacterized SA sensing mechanism. We identified and characterized the novel SA sensing regulator, Rss1, a binuclear zinc cluster protein with dual functions as putative SA receptor and transcriptional activator regulating genes important for SA and tryptophan degradation. Rss1 represents a major component in the identified SA sensing pathway during the fungus’ saprophytic stage. However, Rss1 does not have a detectable impact on virulence. The data presented in this work indicate that alternative or redundant sensing cascades exist that regulate the expression of SA‐responsive genes in U. maydis during its pathogenic development.
PMCID: PMC5082525  PMID: 27387604
15.  Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates 
Molecular microbiology  2014;93(3):505-520.
A remarkable feature of the flagellar-specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook-basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N-terminal secretion signal, mRNA secretion signals within the 5′-untranslated region (5′-UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational β-lactamase fusions to probe the secretion efficacy of the N-terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early-type substrates FlgD, FlgF, FlgE and the late-type substrate FlgL were analysed in detail. Analysing the role of the 5′-UTR in secretion of flgB and flgE revealed that the native 5′-UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate-specific chaperones allowing for targeted secretion of flagellar substrates.
PMCID: PMC5076378  PMID: 24946091
16.  Post-transcriptional regulation by distal Shine-Dalgarno sequences in the grpE-dnaK intergenic region of Streptococcus mutans 
Molecular microbiology  2015;98(2):302-317.
A unique 373 bp region (igr66) between grpE and dnaK of Streptococcus mutans lacks a promoter but is required for optimal production of DnaK. Northern blotting using probes specific to hrcA, igr66 or dnaK revealed multiple transcripts produced from the dnaK operon and 5′-RACE mapped 5′ termini of multiple dnaK transcripts within igr66. One product mapped to a predicted 5′-SL (stem-loop) and two others mapped just 5′ to Shine-Dalgarno (SD)-like sequences located immediately upstream to dnaK and to a predicted SL 120 bp upstream of the dnaK start codon (3′-SL). A collection of cat reporter-gene strains containing mutant derivatives of igr66 were engineered. Chloramphenicol acetyltransferase (CAT) activity varied greatly between strains, but there were no correlative changes in cat mRNA levels. Interestingly, mutations introduced into the SD-like sequences 5′ to the 3′-SL resulted in an 83–98% decrease in CAT activity. Markerless point mutations introduced upstream of dnaK in the SD-like sequences impaired growth at elevated temperatures and resulted in up to a 40% decrease in DnaK protein after heat shock. Collectively, these results indicate processing within igr66 enhances translation in a temperature dependent manner via non-canonical ribosome binding sites positioned > 120 bp upstream of dnaK.
PMCID: PMC4666293  PMID: 26172310
17.  Regulatory Rewiring Confers Serotype-Specific Hyper-Virulence in the Human Pathogen Group A Streptococcus 
Molecular microbiology  2015;98(3):473-489.
Phenotypic heterogeneity is commonly observed between isolates of a given pathogen. Epidemiological analyses have identified that some serotypes of the group A Streptococcus (GAS) are non-randomly associated with particular disease manifestations. Here, we present evidence that a contributing factor to the association of serotype M3 GAS isolates with severe invasive infections is the presence of a null mutant allele for the orphan kinase RocA. Through use of RNAseq analysis, we identified that the natural rocA mutation present within M3 isolates leads to the enhanced expression of more than a dozen immunomodulatory virulence factors, enhancing phenotypes such as hemolysis and NAD+ hydrolysis. Consequently, an M3 GAS isolate survived human phagocytic killing at a level 13-fold higher than a rocA complemented derivative, and was significantly more virulent in a murine bacteremia model of infection. Finally, we identified that RocA functions through the CovR/S two-component system as levels of phosphorylated CovR increase in the presence of functional RocA, and RocA has no regulatory activity following covR or covS mutation. Our data are consistent with RocA interfacing with the CovR/S two-component system, and that the absence of this activity in M3 GAS potentiates the severity of invasive infections caused by isolates of this serotype.
PMCID: PMC4675629  PMID: 26192205
Phenotypic variation; regulation; virulence factors; cross-talk; Streptococcus pyogenes
18.  Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa 
Molecular microbiology  2015;98(2):343-356.
In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically with each other in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, while TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.
PMCID: PMC4690728  PMID: 26193906
19.  Acyl acceptor recognition by Enterococcus faecium l,d-transpeptidase Ldtfm 
Molecular microbiology  2015;98(1):90-100.
In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of β-lactam antibiotics is bypassed by l,d-transpeptidases that form unusual 3→3 peptidoglycan cross-links. β-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l,d-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium l,d-transpeptidase Ldtfm by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An NMR-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldtfm that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilization of the acceptor in the Ldtfm active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β-lactams.
PMCID: PMC4691707  PMID: 26101813
Acyl acceptor; β-lactam; Enterococcus faecium; l,d-transpeptidase; peptidoglycan
20.  CdiA promotes receptor-independent intercellular adhesion 
Molecular microbiology  2015;98(1):175-192.
CdiB/CdiA proteins mediate inter-bacterial competition in a process termed contact-dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI+ cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto-aggregation and biofilm formation in several species, but the contribution of CdiA-receptor interactions to these multi-cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA-BamA mediated cell-cell adhesion. However, CdiA also promotes auto-aggregation in a BamA-independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA-independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA-CdiA interactions. The BamA-dependent and BamA-independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth-inhibition activity. This adhesion should enable “greenbeard” discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind-selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer.
PMCID: PMC4694591  PMID: 26135212
contact-dependent growth inhibition; kind-selection; self/non-self recognition; toxin/immunity proteins; type V secretion systems
21.  Mycobacterium tuberculosis oriC sequestration by MtrA response regulator 
Molecular microbiology  2015;98(3):586-604.
The regulators of Mycobacterium tuberculosis DNA replication are largely unknown. Here, we demonstrate that in synchronously replicating M. tuberculosis, MtrA access to origin of replication (oriC) is enriched in the post-replication (D) period. The increased oriC binding results from elevated MtrA phosphorylation (MtrA~P) as evidenced by reduced expression of dnaN, dnaA and increased expression of select cell division targets. Overproduction of gain-of-function MtrAY102C advanced the MtrA oriC access to the C period, reduced dnaA and dnaN expression, interfered with replication synchrony and compromised cell division. Overproduction of wild-type (MtrA+) or phosphorylation-defective MtrAD56N did not promote oriC access in the C period, nor affected cell cycle progression. MtrA interacts with DnaA signaling a possibility that DnaA helps load MtrA on oriC. Therefore, oriC sequestration by MtrA~P in the D period may normally serve to prevent untimely initiations and that DnaA-MtrA interactions may facilitate regulated oriC replication. Finally, despite the near sequence identity of MtrA in M. smegmatis and M. tuberculosis, the M. smegmatis oriC is not MtrA-target. We conclude that M. tuberculosis oriC has evolved to be regulated by MtrA and that cell cycle progression in this organisms are governed, at least in part, by oscillations in the MtrA~P levels.
PMCID: PMC4700885  PMID: 26207528
Mycobacterium; Cell cycle; Tuberculosis; Response Regulator; Synchronous replication
22.  Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus 
Molecular microbiology  2015;98(2):218-242.
Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activity of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus.
PMCID: PMC4705035  PMID: 26135358
bacillithiol; iron; thiol; iron-sulfur cluster
23.  Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways 
Molecular microbiology  2015;98(2):193-207.
Prokaryotes use subcellular compartments for a variety of purposes. An intriguing example is a family of complex subcellular organelles known as bacterial microcompartments (MCPs). MCPs are widely distributed among bacteria and impact processes ranging global carbon fixation and enteric pathogenesis. Overall, MCPs consist of metabolic enzymes encased within a protein shell, and their function is to optimize biochemical pathways by confining toxic or volatile metabolic intermediates. MCPs are fundamentally different from other organelles in having a complex protein shell rather than a lipid-based membrane as an outer barrier. This unusual feature raises basic questions about organelle assembly, protein targeting and metabolite transport. In this review, we discuss the three best-studied MCPs highlighting atomic-level models for shell assembly, targeting sequences that direct enzyme encapsulation, multivalent proteins that organize the lumen enzymes, the principles of metabolite movement across the shell, internal cofactor recycling, a potential system of allosteric regulation of metabolite transport and the mechanism and rationale behind the functional diversification of the proteins that form the shell. We also touch on some potential biotechnology applications an unusual compartment designed by nature to optimize metabolic processes within a cellular context.
PMCID: PMC4718714  PMID: 26148529
24.  FlhG employs diverse intrinsic domains and influences FlhF GTPase activity to numerically regulate polar flagellar biogenesis in Campylobacter jejuni 
Molecular microbiology  2015;99(2):291-306.
Flagellation in polar flagellates is one of the rare biosynthetic processes known to be numerically regulated in bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. Unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni. Instead, two regions of C. jejuni FlhG that are absent or significantly altered in FlhG orthologs are involved in numerical regulation of flagellar biogenesis. Additionally, we found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. Our work suggests that FlhG ATPases divergently evolved in each polarly flagellated species to employ different intrinsic domains and extrinsic effectors to ultimately mediate a common output – precise numerical control of polar flagellar biogenesis required to create species-specific flagellation patterns optimal for motility.
PMCID: PMC4821507  PMID: 26411371
25.  Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination 
Molecular microbiology  2016;101(4):559-574.
The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription.
PMCID: PMC5038137  PMID: 27125778

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