Bareket Dassa

Splicing of the Mycobacteriophage Bethlehem DnaB Intein: IDENTIFICATION OF A NEW MECHANISTIC CLASS OF INTEINS THAT CONTAIN AN OBLIGATE BLOCK F NUCLEOPHILE*♦

Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala1 inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.

Abundance and Diversity of Dockerin-Containing Proteins in the Fiber-Degrading Rumen Bacterium, Ruminococcus flavefaciens FD-1

Background The cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown. Methodology/Principal Findings We have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca2+-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e.g., cohesin-containing proteins, belonging to the scaffoldins. Conclusions/Significance This report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the parent protein, in response to their role in the rumen environment.

Distribution and function of new bacterial intein-like protein domains.

Hint protein domains appear in inteins and in the C‐terminal region of Hedgehog and Hedgehog‐like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein‐splicing of their host‐protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N‐terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein‐like (BIL) domains. Bacterial intein‐like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C‐terminal flank and could also protein‐splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii

ABSTRACT Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches. Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family (GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to carry dockerin modules, with one, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wall-anchoring protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.” IMPORTANCE Fermentation of dietary nondigestible carbohydrates by the human colonic microbiota supplies much of the energy that supports microbial growth in the intestine. This activity has important consequences for health via modulation of microbiota composition and the physiological and nutritional effects of microbial metabolites, including the supply of energy to the host from short-chain fatty acids. Recent evidence indicates that certain human colonic bacteria play keystone roles in degrading nondigestible substrates, with the dominant but little-studied species Ruminococcus bromii displaying an exceptional ability to degrade dietary resistant starches (i.e., dietary starches that escape digestion by host enzymes in the upper gastrointestinal tract because of protection provided by other polymers, particle structure, retrogradation, or chemical cross-linking). In this report, we reveal the unique organization of the amylolytic enzyme system of R. bromii that involves cohesin-dockerin interactions between component proteins. While dockerins and cohesins are fundamental to the organization of cellulosomal enzyme systems of cellulolytic ruminococci, their contribution to organization of amylases has not previously been recognized and may help to explain the starch-degrading abilities of R. bromii. Fermentation of dietary nondigestible carbohydrates by the human colonic microbiota supplies much of the energy that supports microbial growth in the intestine. This activity has important consequences for health via modulation of microbiota composition and the physiological and nutritional effects of microbial metabolites, including the supply of energy to the host from short-chain fatty acids. Recent evidence indicates that certain human colonic bacteria play keystone roles in degrading nondigestible substrates, with the dominant but little-studied species Ruminococcus bromii displaying an exceptional ability to degrade dietary resistant starches (i.e., dietary starches that escape digestion by host enzymes in the upper gastrointestinal tract because of protection provided by other polymers, particle structure, retrogradation, or chemical cross-linking). In this report, we reveal the unique organization of the amylolytic enzyme system of R. bromii that involves cohesin-dockerin interactions between component proteins. While dockerins and cohesins are fundamental to the organization of cellulosomal enzyme systems of cellulolytic ruminococci, their contribution to organization of amylases has not previously been recognized and may help to explain the starch-degrading abilities of R. bromii.

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.

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

BackgroundMicrobial 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.ResultsComprehensive 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.ConclusionsThis 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.

Cellulosomics of the cellulolytic thermophile Clostridium clariflavum

BackgroundClostridium clariflavum is an anaerobic, thermophilic, Gram-positive bacterium, capable of growth on crystalline cellulose as a single carbon source. The genome of C. clariflavum has been sequenced to completion, and numerous cellulosomal genes were identified, including putative scaffoldin and enzyme subunits.ResultsBioinformatic analysis of the C. clariflavum genome revealed 49 cohesin modules distributed on 13 different scaffoldins and 79 dockerin-containing proteins, suggesting an abundance of putative cellulosome assemblies. The 13-scaffoldin system of C. clariflavum is highly reminiscent of the proposed cellulosome system of Acetivibrio cellulolyticus. Analysis of the C. clariflavum type I dockerin sequences indicated a very high level of conservation, wherein the putative recognition residues are remarkably similar to those of A. cellulolyticus. The numerous interactions among the cellulosomal components were elucidated using a standardized affinity ELISA-based fusion-protein system. The results revealed a rather simplistic recognition pattern of cohesin-dockerin interaction, whereby the type I and type II cohesins generally recognized the dockerins of the same type. The anticipated exception to this rule was the type I dockerin of the ScaB adaptor scaffoldin which bound selectively to the type I cohesins of ScaC and ScaJ.ConclusionsThe findings reveal an intricate picture of predicted cellulosome assemblies in C. clariflavum. The network of cohesin-dockerin pairs provides a thermophilic alternative to those of C. thermocellum and a basis for subsequent utilization of the C. clariflavum cellulosomal system for biotechnological application.

Rumen Cellulosomics: Divergent Fiber-Degrading Strategies Revealed by Comparative Genome-Wide Analysis of Six Ruminococcal Strains

Background A complex community of microorganisms is responsible for efficient plant cell wall digestion by many herbivores, notably the ruminants. Understanding the different fibrolytic mechanisms utilized by these bacteria has been of great interest in agricultural and technological fields, reinforced more recently by current efforts to convert cellulosic biomass to biofuels. Methodology/Principal Findings Here, we have used a bioinformatics-based approach to explore the cellulosome-related components of six genomes from two of the primary fiber-degrading bacteria in the rumen: Ruminococcus flavefaciens (strains FD-1, 007c and 17) and Ruminococcus albus (strains 7, 8 and SY3). The genomes of two of these strains are reported for the first time herein. The data reveal that the three R. flavefaciens strains encode for an elaborate reservoir of cohesin- and dockerin-containing proteins, whereas the three R. albus strains are cohesin-deficient and encode mainly dockerins and a unique family of cell-anchoring carbohydrate-binding modules (family 37). Conclusions/Significance Our comparative genome-wide analysis pinpoints rare and novel strain-specific protein architectures and provides an exhaustive profile of their numerous lignocellulose-degrading enzymes. This work provides blueprints of the divergent cellulolytic systems in these two prominent fibrolytic rumen bacterial species, each of which reflects a distinct mechanistic model for efficient degradation of cellulosic biomass.

Proteome-Based Comparative Analyses of Growth Stages Reveal New Cell Cycle-Dependent Functions in the Predatory Bacterium Bdellovibrio bacteriovorus

ABSTRACT Bdellovibrio and like organisms are obligate predators of bacteria that are ubiquitously found in the environment. Most exhibit a peculiar dimorphic life cycle during which free-swimming attack-phase (AP) cells search for and invade bacterial prey cells. The invader develops in the prey as a filamentous polynucleoid-containing cell that finally splits into progeny cells. Therapeutic and biocontrol applications of Bdellovibrio in human and animal health and plant health, respectively, have been proposed, but more knowledge of this peculiar cell cycle is needed to develop such applications. A proteomic approach was applied to study cell cycle-dependent expression of the Bdellovibrio bacteriovorus proteome in synchronous cultures of a facultative host-independent (HI) strain able to grow in the absence of prey. Results from two-dimensional gel electrophoresis, mass spectrometry, and temporal expression of selected genes in predicted operons were analyzed. In total, about 21% of the in silico predicted proteome was covered. One hundred ninety-six proteins were identified, including 63 hitherto unknown proteins and 140 life stage-dependent spots. Of those, 47 were differentially expressed, including chemotaxis, attachment, growth- and replication-related, cell wall, and regulatory proteins. Novel cell cycle-dependent adhesion, gliding, mechanosensing, signaling, and hydrolytic functions were assigned. The HI model was further studied by comparing HI and wild-type AP cells, revealing that proteins involved in DNA replication and signaling were deregulated in the former. A complementary analysis of the secreted proteome identified 59 polypeptides, including cell contact proteins and hydrolytic enzymes specific to predatory bacteria.

Glycoside hydrolases as components of putative carbohydrate biosensor proteins in Clostridium thermocellum.

The composition of the cellulase system in the cellulosome-producing bacterium, Clostridium thermocellum, has been reported to change in response to growth on different carbon sources. Recently, an extensive carbohydrate-sensing mechanism, purported to regulate the activation of genes coding for polysaccharide-degrading enzymes, was suggested. In this system, CBM modules, comprising extracellular components of RsgI-like anti-σ factors, were proposed to function as carbohydrate sensors, through which a set of cellulose utilization genes are activated by the associated σI-like factors. An extracellular module of one of these RsgI-like proteins (Cthe_2119) was annotated as a family 10 glycoside hydrolase, RsgI6-GH10, and a second putative anti-σ factor (Cthe_1471), related in sequence to Rsi24, was found to contain a module that resembles a family 5 glycoside hydrolase (termed herein Rsi24C-GH5). The present study examines the relevance of these two glycoside hydrolases as sensors in this signal-transmission system. The RsgI6-GH10 was found to bind xylan matrices but exhibited low enzymatic activity on this substrate. In addition, this glycoside hydrolase module was shown to interact with crystalline cellulose although no hydrolytic activity was detected on cellulosic substrates. Bioinformatic analysis of the Rsi24C-GH5 showed a glutamate-to-glutamine substitution that would presumably preclude catalytic activity. Indeed, the recombinant module was shown to bind to cellulose, but showed no hydrolytic activity. These observations suggest that these two glycoside hydrolases underwent an evolutionary adaptation to function as polysaccharide binding agents rather than enzymatic components and thus serve in the capacity of extracellular carbohydrate sensors.