Haft, D. H. | Search bioRxiv

bioRxiv2024

Cyanoexosortase B is essential for motility, biofilm formation and scytonemin production in a filamentous cyanobacterium

Exosortases are involved in trafficking proteins containing PEP-CTERM domains to the exterior of gram-negative bacterial cells. The role of these proteins in cyanobacteria, where such homologs are common, has not been defined. The filamentous cyanobacterium Nostoc punctiforme contains a single putative exosortase, designated cyanoexosortase B (CrtB), implicated by previous work in both the motility, and in the production of the UV-absorbing pigment, scytonemin. To determine the role of crtB in N. punctiforme, a crtB-deletion strain ({Delta}crtB) was generated. {Delta}crtB presented the loss of motility, biofilm formation, and scytonemin production. In the case of motility, the {Delta}crtB mutant exhibited a specific defect in the ability of hormogonia (specialized motile filaments) to adhere to hormogonium polysaccharide (HPS) and several PEP-CTERM proteins expressed in motile hormogonia were differentially abundant in the exoproteome of the wild type compared to the {Delta}crtB strain. These results are consistent with the hypothetical role of CrtB in the processing and export of PEP-CTERM proteins that play a critical role in stabilizing the interaction between the filament surface and HPS to facilitate motility and biofilm formation. In the case of scytonemin, the late biosynthetic steps of which occur in the periplasm and whose operon contains several putative PEP-CTERM proteins, {Delta}crtB failed to produce it. Given the abundance of putative PEP-CTERM proteins encoded in the N. punctiforme genome, and the fact that this study only associates a fraction of them with biological functions, it seems likely that CrtB may play an important role in other biological processes in cyanobacteria. ImportanceIn gram-negative bacteria, exosortases facilitate the trafficking of proteins to the exterior of the cell where they have been implicated in stabilizing the association of extracellular polymeric substances (EPS) with the cell surface to facilitate biofilm formation and flocculation, but the role of exosortases in cyanobacteria has not been explored. Here, we characterize the role of cyanoexosortase B (CrtB) in the filamentous cyanobacterium Nostoc punctiforme, demonstrating that crtB is essential for motility, biofilm formation, and the production of the sunscreen pigment scytonemin. These findings have important implications for understanding motility and biofilm formation in filamentous cyanobacteria as well as efforts toward heterologous production of scytonemin in non-native hosts.

microbiology↗

bioRxiv2023

In silico discovery of the myxosortases that process MYXO-CTERM and three novel prokaryotic C-terminal protein-sorting signals that share invariant Cys residues

The LPXTG protein-sorting signal, found in surface proteins of various Gram-positive pathogens, was the founding member of a growing panel of prokaryotic small C-terminal sorting domains. Sortase A (SrtA) cleaves LPXTG, exosortases (XrtA and XrtB) cleave the PEP-CTERM sorting signal, archaeosortase A (ArtA) cleaves PGF-CTERM, and rhombosortase (RrtA) cleaves GlyGly-CTERM domains. Four sorting signal domains without previously known processing proteases are the MYXO-CTERM, JDVT-CTERM, Synerg-CTERM, and CGP-CTERM domains. These exhibit the standard tripartite architecture of short signature motif, then a hydrophobic transmembrane segment, then an Arg-rich cluster. Each has an invariant cysteine in its signature motif. Computational evidence strongly suggests that each of these four Cys-containing sorting signals is processed, at least in part, by a cognate family of glutamic-type intramembrane endopeptidases, related to eukaryotic type II CAAX-processing protease Rce1. For the MYXO-CTERM sorting signals of different lineages, their sorting enzymes, called myxosortases, include MrtX (MXAN_2755 in Myxococcus xanthus), MrtC, and MrtP, all with radically different N-terminal domains but with a conserved core. Predicted cognate sorting enzymes were identified also for JDVT-CTERM (MrtJ), Synerg-CTERM (MrtS), and CGP-CTERM (MrtA). This work establishes a major new family of protein-sorting housekeeping endopeptidases contributing to surface attachment of proteins in prokaryotes. ImportanceHomologs of the eukaryotic type II CAAX-box protease Rce1, a membrane-embedded endopeptidase found in yeast and human ER and involved in sorting proteins to their proper cellular locations, are abundant in prokaryotes but are not well understood there. This bioinformatics paper identifies several subgroups of the family as cognate endopeptidases for four protein-sorting signals processed by previously unknown machinery. Sorting signals with newly identified processing enzymes include three novel ones, but also MYXO-CTERM, which had been the focus of previous experimental work in the model fruiting and gliding bacterium Myxococcus xanthus. The new findings will substantially improve our understanding of Cys-containing C-terminal protein-sorting signals and of protein trafficking generally in bacteria and archaea.

bioinformatics↗

bioRxiv2023

Myxosortases process MYXO-CTERM and other bacterial C-terminal protein-sorting signals that have invariant Cys residues.

The LPXTG protein-sorting signal, found in surface proteins of various Gram-positive pathogens, was the founding member of a growing panel of prokaryotic small C-terminal sorting domains. Sortase A (SrtA) cleaves LPXTG, exosortases (XrtA and XrtB) cleave the PEP-CTERM sorting signal, archaeosortase A (ArtA) cleaves PGF-CTERM, and rhombosortase (RrtA) cleaves GlyGly-CTERM domains. Three sorting signal domains without previously known processing proteases are the MYXO-CTERM, JDVT-CTERM, and SYNERG-CTERM domains. These exhibit the standard tripartite architecture of short signature motif, then a hydrophobic transmembrane segment, then an Arg-rich cluster. Each has an invariant cysteine in its signature motif. Here, we show computational evidence that these three Cys-containing sorting signals are processed by corresponding subfamilies of glutamic-type intramembrane proteases, related to type II CAAX-processing proteases found in eukaryotes. We name these sorting enzymes generally as myxosortases, and identify MXAN_2755 from Myxococcus xanthus as MrtX (myxosortase X). Additional myxosortases families MrtC and MrtP have radically different N-terminal domains, suggesting most myxosortases act as bifunctional enzymes. Myxosortase-like processing enzymes are identified also for the JDVT-CTERM (MrtJ) and SYNERG-CTERM (MrtS). This work establishes a major new family of protein-sorting housekeeping enzymes for the surface attachment of proteins on bacterial outer membranes.

bioinformatics↗

bioRxiv2022

Eight Unexpected Selenoprotein Families in ABC transport, in Organometallic Biochemistry in Clostridium difficile and other anaerobes, and in Methylmercury Biosynthesis.

A novel protein family related to mercury resistance protein MerB, which cleaves Hg-C bonds of organomercurial compounds, is a newly recognized selenoprotein, typically seen truncated in sequence databases at CU (cysteine-selenocysteine) dipeptide sites fifty residues before the true C-terminus. Inspection shows this protein occurs in a nine-gene neighborhood conserved in more than fifty bacterial species, taxonomically diverse but exclusively anaerobic, including spirochetes, deltaproteobacteria, and Gram-positive spore-formers Clostridium difficile and C. botulinum. Three included families are novel selenoproteins in most instances, including two ABC transporter subunits, one a substrate-binding protein with another CU motif, the other a permease subunit with selenocysteine at the substrate-gating position. Phylogenetic profiling shows a strong pattern of co-occurrence with Stickland metabolism selenoproteins, but an even closer link to a group of 8Fe-9S cofactor-type double-cubane proteins. These 8Fe-9S enzymes vary in count and in genome location but frequently sit next to the nine-gene locus. We have named the locus SAO, because of the Selenocysteine-Assisted Organometallic (SAO) biochemistry implied by an uptake ABC transporter with apparent metal-binding selenocysteines, complementary metal efflux pump SaoE, the MerB-like cytosolic enzyme now called SaoL, and comparative genomics signatures suggesting energy metabolism rather than metal resistance. Hypothesizing cycles of formation and dismutation of organometallic compounds involved in fermentative metabolism, we examined methylmercury formation proteins, and discovered most HgcA proteins are selenoproteins as well, with a CU motif N-terminal to the previously predicted start. Seeking additional rare and overlooked selenoproteins, tricky because of their rarity, could help reveal more candidate cryptic biochemical processes.

microbiology↗

bioRxiv2019

Using the NCBI AMRFinder Tool to Determine Antimicrobial Resistance Genotype-Phenotype Correlations Within a Collection of NARMS Isolates

Antimicrobial resistance (AMR) is a major public health problem that requires publicly available tools for rapid analysis. To identify acquired AMR genes in whole genome sequences, the National Center for Biotechnology Information (NCBI) has produced a high-quality, curated, AMR gene reference database consisting of up-to-date protein and gene nomenclature, a set of hidden Markov models (HMMs), and a curated protein family hierarchy. Currently, the Bacterial Antimicrobial Resistance Reference Gene Database contains 4,579 antimicrobial resistance gene proteins and more than 560 HMMs. Here, we describe AMRFinder, a tool that uses this reference dataset to identify AMR genes. To assess the predictive ability of AMRFinder, we measured the consistency between predicted AMR genotypes from AMRFinder against resistance phenotypes of 6,242 isolates from the National Antimicrobial Resistance Monitoring System (NARMS). This included 5,425 Salmonella enterica, 770 Campylobacter spp., and 47 Escherichia coli phenotypically tested against various antimicrobial agents. Of 87,679 susceptibility tests performed, 98.4% were consistent with predictions. To assess the accuracy of AMRFinder, we compared its gene symbol output with that of a 2017 version of ResFinder, another publicly available resistance gene database. Most gene calls were identical, but there were 1,229 gene symbol differences between them, with differences due to both algorithmic differences and database composition. AMRFinder missed 16 loci that Resfinder found, while Resfinder missed 1,147 loci AMRFinder identified. Two missing drug classes from the 2017 version of ResFinder contributed 81% of missed loci. Based on these results, AMRFinder appears to be a highly accurate AMR gene detection system. ImportanceAntimicrobial resistance is a major public health problem. Traditionally, antimicrobial resistance has been identified using phenotypic assays. With the advent of genome sequencing, we now can identify resistance genes and deduce if an isolate could be resistant to antibiotics. We describe a database of 4,579 acquired antimicrobial resistance genes, the largest publicly available, and a software tool to identify genes in bacterial genomes, AMRFinder. Unlike other tools, AMRFinder uses a gene hierarchy to prevent overpredicting what the correct gene call should be, enabling more accurate assessment. To assess these resources, we determined the resistance gene content of over 6,200 bacterial isolates from the National Antimicrobial Resistance Monitoring System that have been assayed using traditional methods and that also have had their genomes sequenced. We also compared our gene assessments to those of a popularly used tool. We found that AMRFinder has a high overall consistency between genotypes and phenotypes.

microbiology↗