Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence.

The DNA uptake competence (Com) system of the intracellular bacterial pathogen Listeria monocytogenes is considered nonfunctional. There are no known conditions for DNA transformation, and the Com master activator gene, comK, is interrupted by a temperate prophage. Here, we show that the L. monocytogenes Com system is required during infection to promote bacterial escape from macrophage phagosomes in a manner that is independent of DNA uptake. Further, we find that regulation of the Com system relies on the formation of a functional comK gene via prophage excision. Prophage excision is specifically induced during intracellular growth, primarily within phagosomes, yet, in contrast to classic prophage induction, progeny virions are not produced. This study presents the characterization of an active prophage that serves as a genetic switch to modulate the virulence of its bacterial host in the course of infection.

A GC-Rich Prophage-Like Genomic Region of Mycoplasma bovirhinis HAZ141_2 Carries a Gene Cluster Encoding Resistance to Kanamycin and Neomycin.

Recently, a complete genome sequence of Mycoplasma bovirhinis HAZ141_2 was published showing the presence of a 54-kB prophage-like region. Bioinformatic analysis revealed that this region has a more than 40% GC content and a chimeric organization with three structural elements-a prophage continuous region, a restriction-modification cassette, and a highly transmittable aadE-sat4-aphA-3 gene cluster found in both Gram-positive and Gram-negative bacteria. It is known that aadE confers resistance to streptomycin, sat4 governs resistance to streptothricin/nourseothricin, and aphA-3 is responsible for resistance to kanamycin and structurally related antibiotics. An aadE-like (aadE*) gene of strain HAZ141_2 encodes a 228-amino acid (aa) polypeptide whose carboxy-terminal domain (positions 44 to 206) is almost identical to that of a functional 302-aa AadE (positions 140 to 302). Transcription analysis of the aadE*-sat4-aphA-3 genes showed their cotranscription in M. bovirhinis HAZ141_2. Moreover, a common promoter for aadE*-sat4-aphA-3 was mapped upstream of aadE* using 5' rapid amplification of cDNA ends analysis. Determination of MICs to aminoglycosides and nourseothricin revealed that M. bovirhinis HAZ141_2 is highly resistant to kanamycin and neomycin (≥512 µg/ml). However, MICs to streptomycin (64 µg/ml) and nourseothricin (16 to 32 µg/ml) were similar to those identified in the prophageless M. bovirhinis type strain PG43 and Israeli field isolate 316981. We cloned the aadE*-sat4-aphA-3 genes into a low-copy-number vector and transferred them into antibiotic-sensitive Escherichia coli cells. While the obtained E. coli transformants were highly resistant to kanamycin, neomycin, and nourseothricin (MICs, ≥256 µg/ml), there were no changes in MICs to streptomycin, suggesting a functional defect of the aadE*.

Controlled branched-chain amino acids auxotrophy in Listeria monocytogenes allows isoleucine to serve as a host signal and virulence effector.

Listeria monocytogenes (Lm) is a saprophyte and intracellular pathogen. Transition to the pathogenic state relies on sensing of host-derived metabolites, yet it remains unclear how these are recognized and how they mediate virulence gene regulation. We previously found that low availability of isoleucine signals Lm to activate the virulent state. This response is dependent on CodY, a global regulator and isoleucine sensor. Isoleucine-bound CodY represses metabolic pathways including branched-chain amino acids (BCAA) biosynthesis, however under BCAA depletion, as occurs during infection, BCAA biosynthesis is upregulated and isoleucine-unbound CodY activates virulence genes. While isoleucine was revealed as an important input signal, it was not identified how internal levels are controlled during infection. Here we show that Lm regulates BCAA biosynthesis via CodY and via a riboregulator located upstream to the BCAA biosynthesis genes, named Rli60. rli60 is transcribed when BCAA levels drop, forming a ribosome-mediated attenuator that cis-regulates the downstream genes according to BCAA supply. Notably, we found that Rli60 restricts BCAA production, essentially starving Lm, a mechanism that is directly linked to virulence, as it controls the internal isoleucine pool and thereby CodY activity. This controlled BCAA auxotrophy likely evolved to enable isoleucine to serve as a host signal and virulence effector.

Genomics-based epidemiology of bovine Mycoplasma bovis strains in Israel.

BACKGROUND: Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis affecting cattle production and animal welfare. In the past in Israel, M. bovis has been most frequently associated with bovine respiratory disease (BRD) and was rarely isolated from mastitis. This situation changed in 2008 when M. bovis-associated mastitis emerged in Israel. The aim of this study was to utilize whole genome sequencing to evaluate the molecular epidemiology and genomic diversity of M. bovis mastitis-associated strains and their genetic relatedness to M. bovis strains isolated from BRD in local feedlot calves and those imported to Israel from different European countries and Australia. RESULTS: Phylogeny based on total single nucleotide polymorphism (SNP) analysis of 225 M. bovis genomes clearly showed clustering of isolates on the basis of geographical origin: strains isolated from European countries clustered together and separately from Australian and Chinese isolates, while Israeli isolates were found in the both groups. The dominant genotype was identified among local mastitis-associated M. bovis isolates. This genotype showed a close genomic relatedness to M. bovis strains isolated from calves imported to Israel from Australia, to original Australian M. bovis strains, as well as to strains isolated in China. CONCLUSIONS: This study represents the first comprehensive high-resolution genome-based epidemiological analysis of M. bovis in Israel and illustrates the possible dissemination of the pathogen across the globe by cattle trade.

Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum.

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme cellulosome complex, certain clostridia contain alternative sigma I (σI ) factors that have cognate membrane-associated anti-σI factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure-function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14-superfamily motifs. The X-ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σI4 -dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σI3 -dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation-related genes. Structural similarity between clostridial PA14 dyads to PA14-containing proteins in yeast helped identify another crucial signature element: the calcium-binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14A ) is dominant in directing the binding to the ligand in both bacteria. The two X-ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.

Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum.

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σI factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum In the present report, we provide experimental evidence demonstrating that the C. thermocellum cipA gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σI factors and by the vegetative σA factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of C. thermocellum cipA are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σI- and σA-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, Clostridium straminisolvens, Clostridium clariflavum, Acetivibrio cellulolyticus, and Clostridium sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of Clostridium cellulovorans matches the predicted σI-dependent promoter identified in the present work rather than the previously proposed σA promoter. With the exception of C. cellulovorans, both σI and σA promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components.IMPORTANCE Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of C. thermocellum and related cellulosome-producing bacteria en route to the consolidated bioprocessing of biomass.

An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus.

UNLABELLED: Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. IMPORTANCE: In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA.

Metabolic adaptations are critical to the ability of bacterial pathogens to grow within host cells and are normally preceded by sensing of host-specific metabolic signals, which in turn can influence the pathogen's virulence state. Previously, we reported that the intracellular bacterial pathogen Listeria monocytogenes responds to low availability of branched-chain amino acids (BCAAs) within mammalian cells by up-regulating both BCAA biosynthesis and virulence genes. The induction of virulence genes required the BCAA-responsive transcription regulator, CodY, but the molecular mechanism governing this mode of regulation was unclear. In this report, we demonstrate that CodY directly binds the coding sequence of the L. monocytogenes master virulence activator gene, prfA, 15 nt downstream of its start codon, and that this binding results in up-regulation of prfA transcription specifically under low concentrations of BCAA. Mutating this site abolished CodY binding and reduced prfA transcription in macrophages, and attenuated bacterial virulence in mice. Notably, the mutated binding site did not alter prfA transcription or PrfA activity under other conditions that are known to activate PrfA, such as during growth in the presence of glucose-1-phosphate. This study highlights the tight crosstalk between L. monocytogenes metabolism and virulence, while revealing novel features of CodY-mediated regulation.

High Prevalence of Diverse Insertion Sequences within the rRNA Operons of Mycoplasma bovis.

Insertion sequences (ISs) are widespread in the genome of Mycoplasma bovis strain PG45, but no ISs were identified within its two tandemly positioned rRNA operons (rrn1 and rrn2). However, characterization of the rrn locus in 70 M. bovis isolates revealed the presence of ISs related to the ISMbov1 (IS30 family) and ISMbov4 (IS4 family) isomers in 35 isolates. ISs were inserted into intergenic region 1 (IGR-1) or IGR-3, which are the putative promoter regions of rrn1 and rrn2, respectively, and into IGR-5, located downstream of the rrl2 gene. Seven different configurations (A to G) of the rrn locus with respect to ISs were identified, including those in five annotated genomes. The transcriptional start site for the single rrn operon in M. bovis strain 88127 was mapped within IGR-1, 60 bp upstream of the rrs gene. Notably, only 1 nucleotide separated the direct repeat (DR) for ISMbov1 and the promoter -35 element in configuration D, while in configuration F, the -35 motif was a part of the ISMbov1 DR. Relative quantitative real-time (qRT) PCR analysis and growth rate comparisons detected a significant increase (P < 0.05) in the expression of the rrs genes and in the number of viable cells during log phase growth (8, 12, and 16 h) in the strains with configuration F in comparison to strains with one or two rrn operons that did not have ISs. A high prevalence of IS elements within or close to the M. bovis rrn operon-promoter region may reflect their important role in regulation of both ribosome synthesis and function. IMPORTANCE: Data presented in this study show a high prevalence of diverse ISs within the M. bovis rrn locus resulting in intraspecies variability and diversity. Such abundance of IS elements near or within the rrn locus may offer a selective advantage to M. bovis Moreover, the fact that expression of the rrs genes as well as the number of viable cells increased in the group of strains with IS element insertion within a putative promoter -35 sequence (configuration F) in comparison to that in strains with one or two rrn operons that do not have ISs may serve as a basis for understanding the possible role of M. bovis IS elements in fundamental biological processes such as regulation of ribosome synthesis and function.

Ruminococcal cellulosome systems from rumen to human.

A cellulolytic fiber-degrading bacterium, Ruminococcus champanellensis, was isolated from human faecal samples, and its genome was recently sequenced. Bioinformatic analysis of the R. champanellensis genome revealed numerous cohesin and dockerin modules, the basic elements of the cellulosome, and manual sequencing of partially sequenced genomic segments revealed two large tandem scaffoldin-coding genes that form part of a gene cluster. Representative R. champanellensis dockerins were tested against putative cohesins, and the results revealed three different cohesin-dockerin binding profiles which implied two major types of cellulosome architectures: (i) an intricate cell-bound system and (ii) a simplistic cell-free system composed of a single cohesin-containing scaffoldin. The cell-bound system can adopt various enzymatic architectures, ranging from a single enzyme to a large enzymatic complex comprising up to 11 enzymes. The variety of cellulosomal components together with adaptor proteins may infer a very tight regulation of its components. The cellulosome system of the human gut bacterium R. champanellensis closely resembles that of the bovine rumen bacterium Ruminococcus flavefaciens. The two species contain orthologous gene clusters comprising fundamental components of cellulosome architecture. Since R. champanellensis is the only human colonic bacterium known to degrade crystalline cellulose, it may thus represent a keystone species in the human gut.

Integrative genomic analysis identifies isoleucine and CodY as regulators of Listeria monocytogenes virulence.

Intracellular bacterial pathogens are metabolically adapted to grow within mammalian cells. While these adaptations are fundamental to the ability to cause disease, we know little about the relationship between the pathogen's metabolism and virulence. Here we used an integrative Metabolic Analysis Tool that combines transcriptome data with genome-scale metabolic models to define the metabolic requirements of Listeria monocytogenes during infection. Twelve metabolic pathways were identified as differentially active during L. monocytogenes growth in macrophage cells. Intracellular replication requires de novo synthesis of histidine, arginine, purine, and branch chain amino acids (BCAAs), as well as catabolism of L-rhamnose and glycerol. The importance of each metabolic pathway during infection was confirmed by generation of gene knockout mutants in the respective pathways. Next, we investigated the association of these metabolic requirements in the regulation of L. monocytogenes virulence. Here we show that limiting BCAA concentrations, primarily isoleucine, results in robust induction of the master virulence activator gene, prfA, and the PrfA-regulated genes. This response was specific and required the nutrient responsive regulator CodY, which is known to bind isoleucine. Further analysis demonstrated that CodY is involved in prfA regulation, playing a role in prfA activation under limiting conditions of BCAAs. This study evidences an additional regulatory mechanism underlying L. monocytogenes virulence, placing CodY at the crossroads of metabolism and virulence.

Fine-structural variance of family 3 carbohydrate-binding modules as extracellular biomass-sensing components of Clostridium thermocellum anti-σI factors.

The anaerobic, thermophilic, cellulosome-producing bacterium Clostridium thermocellum relies on a variety of carbohydrate-active enzymes in order to efficiently break down complex carbohydrates into utilizable simple sugars. The regulation mechanism of the cellulosomal genes was unknown until recently, when genomic analysis revealed a set of putative operons in C. thermocellum that encode σI factors (i.e. alternative σ factors that control specialized regulon activation) and their cognate anti-σI factor (RsgI). These putative anti-σI-factor proteins have modules that are believed to be carbohydrate sensors. Three of these modules were crystallized and their three-dimensional structures were solved. The structures show a high overall degree of sequence and structural similarity to the cellulosomal family 3 carbohydrate-binding modules (CBM3s). The structures of the three carbohydrate sensors (RsgI-CBM3s) and a reference CBM3 are compared in the context of the structural determinants for the specificity of cellulose and complex carbohydrate binding. Fine structural variations among the RsgI-CBM3s appear to result in alternative substrate preferences for each of the sensors.

Distribution and infection-related functions of bacillithiol in Staphylococcus aureus.

Bacillithiol (Cys-GlcN-malate, BSH) serves as a major low molecular weight thiol in low GC Gram-positive bacteria including Bacillus species and a variety of Staphylococcus aureus strains. These bacteria do not produce glutathione (GSH). In this study, HPLC analyses were used to determine BSH levels in different S. aureus strains. Furthermore, the role of BSH in the resistance against oxidants and antibiotics and its function in virulence was investigated. We and others (Newton, G.L., Fahey, R.C., Rawat, M., 2012. Microbiology 158, 1117-1126) found that BSH is not produced by members of the S. aureus NCTC8325 lineage, such as strains 8325-4 and SH1000. Using bioinformatics we show that the BSH-biosynthetic gene bshC is disrupted by an 8-bp duplication in S. aureus NCTC8325. The functional bshC-gene from BSH-producing S. aureus Newman (NWMN_1087) was expressed in S. aureus 8325-4 to reconstitute BSH-synthesis. Comparison of the BSH-producing and BSH-minus strains revealed higher resistance of the BSH-producing strain against the antibiotic fosfomycin and the oxidant hypochlorite but not against hydrogen peroxide or diamide. In addition, a higher bacterial load of the BSH-producing strain was detected in human upper-airway epithelial cells and murine macrophages. This indicates a potential role of BSH in protection of S. aureus during infection.

Structure of a family 3a carbohydrate-binding module from the cellulosomal scaffoldin CipA of Clostridium thermocellum with flanking linkers: implications for cellulosome structure.

The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 Å. The structure is a ß-sandwich with a structural Ca(2+) ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly.

Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors.

Clostridium thermocellum produces a highly efficient cellulolytic extracellular complex, termed the cellulosome, for hydrolyzing plant cell wall biomass. The composition of the cellulosome is affected by the presence of extracellular polysaccharides; however, the regulatory mechanism is unknown. Recently, we have identified in C. thermocellum a set of putative σ and anti-σ factors that include extracellular polysaccharide-sensing components [Kahel-Raifer et al. (2010) FEMS Microbiol Lett 308:84-93]. These factor-encoding genes are homologous to the Bacillus subtilis bicistronic operon sigI-rsgI, which encodes for an alternative σ(I) factor and its cognate anti-σ(I) regulator RsgI that is functionally regulated by an extracytoplasmic signal. In this study, the binding of C. thermocellum putative anti-σ(I) factors to their corresponding σ factors was measured, demonstrating binding specificity and dissociation constants in the range of 0.02 to 1 µM. Quantitative real-time RT-PCR measurements revealed three- to 30-fold up-expression of the alternative σ factor genes in the presence of cellulose and xylan, thus connecting their expression to direct detection of their extracellular polysaccharide substrates. Cellulosomal genes that are putatively regulated by two of these σ factors, σ(I1) or σ(I6), were identified based on the sequence similarity of their promoters. The ability of σ(I1) to direct transcription from the sigI1 promoter and from the promoter of celS (encodes the family 48 cellulase) was demonstrated in vitro by runoff transcription assays. Taken together, the results reveal a regulatory mechanism in which alternative σ factors are involved in regulating the cellulosomal genes via an external carbohydrate-sensing mechanism.

Draft genome sequences for Clostridium thermocellum wild-type strain YS and derived cellulose adhesion-defective mutant strain AD2.

Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain, AD2, played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

Genome-wide analysis of acetivibrio cellulolyticus provides a blueprint of an elaborate cellulosome system.

BACKGROUND: Microbial degradation of plant cell walls and its conversion to sugars and other byproducts is a key step in the carbon cycle on Earth. In order to process heterogeneous plant-derived biomass, specialized anaerobic bacteria use an elaborate multi-enzyme cellulosome complex to synergistically deconstruct cellulosic substrates. The cellulosome was first discovered in the cellulolytic thermophile, Clostridium thermocellum, and much of our knowledge of this intriguing type of protein composite is based on the cellulosome of this environmentally and biotechnologically important bacterium. The recently sequenced genome of the cellulolytic mesophile, Acetivibrio cellulolyticus, allows detailed comparison of the cellulosomes of these two select cellulosome-producing bacteria. RESULTS: Comprehensive analysis of the A. cellulolyticus draft genome sequence revealed a very sophisticated cellulosome system. Compared to C. thermocellum, the cellulosomal architecture of A. cellulolyticus is much more extensive, whereby the genome encodes for twice the number of cohesin- and dockerin-containing proteins. The A. cellulolyticus genome has thus evolved an inflated number of 143 dockerin-containing genes, coding for multimodular proteins with distinctive catalytic and carbohydrate-binding modules that play critical roles in biomass degradation. Additionally, 41 putative cohesin modules distributed in 16 different scaffoldin proteins were identified in the genome, representing a broader diversity and modularity than those of Clostridium thermocellum. Although many of the A. cellulolyticus scaffoldins appear in unconventional modular combinations, elements of the basic structural scaffoldins are maintained in both species. In addition, both species exhibit similarly elaborate cell-anchoring and cellulosome-related gene- regulatory elements. CONCLUSIONS: This work portrays a particularly intricate, cell-surface cellulosome system in A. cellulolyticus and provides a blueprint for examining the specific roles of the various cellulosomal components in the degradation of complex carbohydrate substrates of the plant cell wall by the bacterium.

Listeria monocytogenes TcyKLMN Cystine/Cysteine Transporter Facilitates Glutathione Synthesis and Virulence Gene Expression.

Listeria monocytogenes is a saprophyte and a human intracellular pathogen. Upon invasion into mammalian cells, it senses multiple metabolic and environmental signals that collectively trigger its transition to the pathogenic state. One of these signals is the tripeptide glutathione, which acts as an allosteric activator of L. monocytogenes's master virulence regulator, PrfA. While glutathione synthesis by L. monocytogenes was shown to be critical for PrfA activation and virulence gene expression, it remains unclear how this tripeptide is synthesized in changing environments, especially in light of the observation that L. monocytogenes is auxotrophic to one of its precursors, cysteine. Here, we show that the ABC transporter TcyKLMN is a cystine/cysteine importer that supplies cysteine for glutathione synthesis, hence mediating the induction of the virulence genes. Further, we demonstrate that this transporter is negatively regulated by three metabolic regulators, CodY, CymR, and CysK, which sense and respond to changing concentrations of branched-chain amino acids (BCAA) and cysteine. The data indicate that under low concentrations of BCAA, TcyKLMN is upregulated, driving the production of glutathione by supplying cysteine, thereby facilitating PrfA activation. These findings provide molecular insight into the coupling of L. monocytogenes metabolism and virulence, connecting BCAA sensing to cysteine uptake and glutathione biosynthesis as a mechanism that controls virulence gene expression. This study exemplifies how bacterial pathogens sense their intracellular environment and exploit essential metabolites as effectors of virulence. IMPORTANCE Bacterial pathogens sense the repertoire of metabolites in the mammalian niche and use this information to shift into the pathogenic state to accomplish a successful infection. Glutathione is a virulence-activating signal that is synthesized by L. monocytogenes during infection of mammalian cells. In this study, we show that cysteine uptake via TcyKLMN drives glutathione synthesis and virulence gene expression. The data emphasize the intimate cross-regulation between metabolism and virulence in bacterial pathogens.

A dual-function phage regulator controls the response of cohabiting phage elements via regulation of the bacterial SOS response.

Listeria monocytogenes strain 10403S harbors two phage elements in its chromosome; one produces infective virions and the other tailocins. It was previously demonstrated that induction of the two elements is coordinated, as they are regulated by the same anti-repressor. In this study, we identified AriS as another phage regulator that controls the two elements, bearing the capacity to inhibit their lytic induction under SOS conditions. AriS is a two-domain protein that possesses two distinct activities, one regulating the genes of its encoding phage and the other downregulating the bacterial SOS response. While the first activity associates with the AriS N-terminal AntA/AntB domain, the second associates with its C-terminal ANT/KilAC domain. The ANT/KilAC domain is conserved in many AriS-like proteins of listerial and non-listerial prophages, suggesting that temperate phages acquired such dual-function regulators to align their response with the other phage elements that cohabit the genome.

Implications of the inability of Listeria monocytogenes EGD-e to grow anaerobically due to a deletion in the class III NrdD ribonucleotide reductase for its use as a model laboratory strain.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that causes life-threatening diseases in humans. It grows and survives in environments of low oxygen tension and under conditions of strict anaerobiosis. Oxygen-limiting conditions may be an important factor in determining its pathogenicity. L. monocytogenes serovar 1/2a strain EGD-e has been employed intensively to elucidate the mechanisms of intracellular multiplication and virulence. Listeria possesses genes encoding class I aerobic and class III anaerobic ribonucleotide reductases (RNRs). The class III RNR consists of a catalytic subunit NrdD and an activase NrdG. Surprisingly, L. monocytogenes EGD-e, but not other L. monocytogenes strains or other listerial species, is unable to grow under strict anaerobic conditions. Inspection of listerial NrdD amino acid sequences revealed a six-amino acid deletion in the C-terminal portion of the EGD-e protein, next to the essential glycyl radical domain. Nevertheless, L. monocytogenes EGD-e can grow under microaerophilic conditions due to the recruitment of residual class Ia RNR activity. A three-dimensional (3D) model based on the structure of bacteriophage T4 NrdD identified the location of the deletion, which appears in a highly conserved part of the NrdD RNR structure, in the α/ß barrel domain near the glycyl radical domain. The deleted KITPFE region is essential either for interactions with the NrdG activase or, indirectly, for the stability of the glycyl radical loop. Given that L. monocytogenes EGD-e lacks a functional anaerobic RNR, the present findings are relevant to the interpretation of studies of pathogenesis with this strain specifically, in particular under conditions of low oxygen tension.