The hmsT 3' untranslated region mediates c-di-GMP metabolism and biofilm formation in Yersinia pestis.

Yersinia pestis, the cause of plague, forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is regulated by the intracellular levels of cyclic diguanylate (c-di-GMP). In this study, we investigated the role of the 3' untranslated region (3'UTR) in hmsT mRNA, a transcript that encodes a diguanylate cyclase that stimulates biofilm formation in Y. pestis by synthesizing the second messenger c-di-GMP. Deletion of the 3'UTR increased the half-life of hmsT mRNA, thereby upregulating c-di-GMP levels and biofilm formation. Our findings indicate that multiple regulatory sequences might be present in the hmsT 3'UTR that function together to mediate mRNA turnover. We also found that polynucleotide phosphorylase is partially responsible for hmsT 3'UTR-mediated mRNA decay. In addition, the hmsT 3'UTR strongly repressed gene expression at 37°C and 26°C, but affected gene expression only slightly at 21°C. Our findings suggest that the 3'UTR might be involved in precise and rapid regulation of hmsT expression, allowing Y. pestis to fine-tune c-di-GMP synthesis and consequently regulate biofilm production to adapt to the changing host environment.

CRISPR-Cas12a-Assisted Recombineering in Bacteria.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria.IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

Ecological Opportunity, Evolution, and the Emergence of Flea-Borne Plague.

The plague bacillus Yersinia pestis is unique among the pathogenic Enterobacteriaceae in utilizing an arthropod-borne transmission route. Transmission by fleabite is a recent evolutionary adaptation that followed the divergence of Y. pestis from the closely related food- and waterborne enteric pathogen Yersinia pseudotuberculosis A combination of population genetics, comparative genomics, and investigations of Yersinia-flea interactions have disclosed the important steps in the evolution and emergence of Y. pestis as a flea-borne pathogen. Only a few genetic changes, representing both gene gain by lateral transfer and gene loss by loss-of-function mutation (pseudogenization), were fundamental to this process. The emergence of Y. pestis fits evolutionary theories that emphasize ecological opportunity in adaptive diversification and rapid emergence of new species.

HmsC, a periplasmic protein, controls biofilm formation via repression of HmsD, a diguanylate cyclase in Yersinia pestis.

Yersinia pestis, the cause of plague, forms a biofilm in the foregut of its flea vector to enhance transmission. Biofilm formation in Y. pestis is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP). HmsT and Y3730, the two diguanylate cyclases (DGC) in Y. pestis, are responsible for the synthesis of c-di-GMP. Y3730, which we name here as HmsD, has little effect on in vitro biofilms, but has a major effect on biofilm formation in the flea. The mechanism by which HmsD plays differential roles in vivo and in vitro is not understood. In this study, we show that hmsD is part of a three-gene operon (y3729-31), which we designate as hmsCDE. Deletion of hmsC resulted in increased, hmsD-dependent biofilm formation, while deletion or overexpression of hmsE did not affect biofilm formation. Localization experiments suggest that HmsC resides in the periplasmic space. In addition, we provide evidence that HmsC might interact directly with the periplasmic domain of HmsD and cause the proteolysis of HmsD. We propose that HmsC senses the environmental signals, which in turn regulates HmsD, and controls the c-di-GMP synthesis and biofilm formation in Y. pestis.

Identification of Toxic Proteins Encoded by Mycobacteriophage TM4 Using a Next-Generation Sequencing-Based Method.

Mycobacteriophages are viruses that specifically infect mycobacteria and which, due to their diversity, represent a large gene pool. Characterization of the function of these genes should provide useful insights into host-phage interactions. Here, we describe a next-generation sequencing (NGS)-based, high-throughput screening approach for the identification of mycobacteriophage-encoded proteins that are toxic to mycobacteria. A plasmid-derived library representing the mycobacteriophage TM4 genome was constructed and transformed into Mycobacterium smegmatis. NGS and growth assays showed that the expression of TM4 gp43, gp77, -78, and -79, or gp85 was toxic to M. smegmatis. Although the genes associated with bacterial toxicity were expressed during phage infection, they were not required for lytic replication of mycobacteriophage TM4. In conclusion, we describe here an NGS-based approach which required significantly less time and resources than traditional methods and allowed the identification of novel mycobacteriophage gene products that are toxic to mycobacteria. IMPORTANCE The wide spread of drug-resistant Mycobacterium tuberculosis has brought an urgent need for new drug development. Mycobacteriophages are natural killers of M. tuberculosis, and their toxic gene products might provide potential anti-M. tuberculosis candidates. However, the enormous genetic diversity of mycobacteriophages poses challenges for the identification of these genes. Here, we used a simple and convenient screening method, based on next-generation sequencing, to identify mycobacteriophage genes encoding toxic products for mycobacteria. Using this approach, we screened and validated several toxic products encoded by mycobacteriophage TM4. In addition, we also found that the genes encoding these toxic products are nonessential for lytic replication of TM4. Our work describes a promising method for the identification of phage genes that encode proteins that are toxic to mycobacteria and which might facilitate the identification of novel antimicrobial molecules.

A frameshift in Yersinia pestis rcsD alters canonical Rcs signalling to preserve flea-mammal plague transmission cycles.

Multiple genetic changes in the enteric pathogen Yersinia pseudotuberculosis have driven the emergence of Yesinia pestis, the arthropod-borne, etiological agent of plague. These include developing the capacity for biofilm-dependent blockage of the flea foregut to enable transmission by flea bite. Previously, we showed that pseudogenization of rcsA, encoding a component of the Rcs signalling pathway, is an important evolutionary step facilitating Y. pestis flea-borne transmission. Additionally, rcsD, another important gene in the Rcs system, harbours a frameshift mutation. Here, we demonstrated that this rcsD mutation resulted in production of a small protein composing the C-terminal RcsD histidine-phosphotransferase domain (designated RcsD-Hpt) and full-length RcsD. Genetic analysis revealed that the rcsD frameshift mutation followed the emergence of rcsA pseudogenization. It further altered the canonical Rcs phosphorylation signal cascade, fine-tuning biofilm production to be conducive with retention of the pgm locus in modern lineages of Y. pestis. Taken together, our findings suggest that a frameshift mutation in rcsD is an important evolutionary step that fine-tuned biofilm production to ensure perpetuation of flea-mammal plague transmission cycles.

Yersinia pestis, the agent responsible for the plague, emerged 6,000 to 7,000 years ago from Yersinia pseudotuberculosis, another type of bacteria which still exists today. Although they are highly similar genetically, these two species are strikingly different. While Y. pseudotuberculosis spreads via food and water and causes mild stomach distress, Y. pestis uses fleas to infect new hosts and has killed millions. A small set of genetic changes has contributed to the emergence of Y. pestis by allowing it to thrive inside a flea and maximise its transmission. In particular, some of these mutations have led to the bacteria being able to come together to form a sticky layer that adheres to the gut of the insect, with this 'biofilm' stopping the flea from feeding on blood. The starving flea keeps trying to feed, and with each bite comes another opportunity for Y. pestis to jump host. However, it remains unclear exactly how the mutations have influenced biofilm formation to allow for this new transmission mechanism to take place. To examine this phenomenon, Guo et al. focused on rcsD, a gene that codes for a component of the signalling system that controls biofilm creation. In Y. pestis this sequence has been mutated to become a 'pseudogene', a type of sequence which is often thought to be non-functional. However, the experiments showed that, in Y. pestis, rcsD could produce small amounts of a full-length RcsD protein similar to the one found in Y. pseudotuberculosis. However, the gene mostly produces a short 'RcsD-Hpt' protein that can, in turn, alter the expression of many genes, including those that decrease biofilm formation. This may prove to be beneficial for Y. pestis, for example when the bacteria switches from living in fleas to living in humans, where it does not require a biofilm. Guo et al. further investigated the impact of rcsD becoming a pseudogene inY. pestis, showing that if normal amounts of the full-length RcsD protein are produced, the bacteria quickly lose the gene that allows them to form biofilm in fleas, and cause disease in humans. In fact, additional analyses revealed that all sequenced strains of ancient and modern Y. pestis bacteria can produce RcsD-Hpt, even if they do not carry the same exact rcsD mutation. Overall, these results indicate that rcsD turning into a pseudogene marked an important step in the emergence of Y. pestis strains that can cause lasting plague outbreaks. They also point towards pseudogenes having more important roles in evolution than previously thought.

The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT.

Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT.

Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.

CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.

Mining of the Novel Virulent ATP-Binding Cassette Importers in Mycobacterium abscessus by Comparative Genomic Strategy.

Background: The virulent ATP-binding cassette (ABC) importers from Mycobacterium abscessus, the most common native multidrug resistant and emerging opportunistic pathogen in rapidly growing NTM, were explored by comparative genomic study, in view of the fact that the ABC importers of Mycobacterium tuberculosis, responsible for uptaking metals, anions, amino acids, peptides, sugars, and other crucial substances from the host, had been proved to be closely related with the bacillus's virulence, survival in the host macrophages, antibiotic resistance, modulation of host immune system, and so on, although detailed mechanism was unclear. Methods: For virulent ABC importers from M. abscessus predicted by orthology and phylogeny analysis of nucleotide-binding domains (NBDs) of Mycobacterium smegmatis, M. abscessus, and M. tuberculosis, the antibiotic susceptibility of overexpression transformant and knockout mutant was assayed after confirmation by in vitro experiment. Results: Three-domain importers were dominant ones in M. abscessus (60.0%), four-domain ones dominant in M. tuberculosis (87.5%), whereas both types were same in M. smegmatis (41.9%). In the phylogenetic tree, the importers of M. abscessus (53.3%) and M. tuberculosis (62.5%) were mainly distributed in clay A, whereas the clay E was exclusively composed of M. smegmatis NBDs, which hinted possible reprogramming of the transporter system during the pathogen evolution. In clay A, MAB_2178 and others were predicted virulence-associated because of high sequence similarity to M. tuberculosis virulence importers. Conclusions: The importance and complexity of antibiotics resistance mechanisms of MAB_2176-2177-2178 were pointed out by its overexpression enhancing bacterial resistance to ciprofloxacin, clarithromycin, cefoxitin, and sensitivity to amikacin, and knockout having opposite phenotypes.

Toxin-Antitoxin Systems Alter Adaptation of Mycobacterium smegmatis to Environmental Stress.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

Experimental evidence for negative selection in the evolution of a Yersinia pestis pseudogene.

Yersinia pestis, the agent of bubonic plague, evolved from the enteric pathogen Yersinia pseudotuberculosis within the past 20,000 years. Because ancestor and descendant both exist, it is possible to infer steps in molecular evolution by direct experimental approaches. The Y. pestis life cycle includes establishment of a biofilm within its vector, the flea. Although Y. pseudotuberculosis makes biofilms in other environments, it fails to do so in the insect. We show that rcsA, a negative regulator of biofilms that is functional in Y. pseudotuberculosis, is a pseudogene in Y. pestis. Replacement of the pseudogene with the functional Y. pseudotuberculosis rcsA allele strongly represses biofilm formation and essentially abolishes flea biofilms. The conversion of rcsA to a pseudogene during Y. pestis evolution, therefore, was a case of negative selection rather than neutral genetic drift.

Type VII Toxin/Antitoxin Classification System for Antitoxins that Enzymatically Neutralize Toxins.

Toxin/antitoxin (TA) systems are present in nearly all bacterial and archaeal strains and consist of a toxin that reduces growth and an antitoxin that masks toxin activity. Currently there are six primary classes for TA systems based on the nature of the antitoxin and the way that the antitoxin inactivates the toxin. Here we show that there now are at least three additional and distinct TA systems in which the antitoxin is an enzyme and the cognate toxin is the direct target of the antitoxin: Hha/TomB (antitoxin oxidizes Cys18 of the toxin), TglT/TakA (antitoxin phosphorylates Ser78 of the toxin), and HepT/MntA (antitoxin adds three AMPs to Tyr104 of the toxin). Thus, we suggest the type VII TA system should be used to designate those TA systems in which the enzyme antitoxin chemically modifies the toxin post-translationally to neutralize it. Defining the type VII TA system using this specific criterion will aid researchers in classifying newly discovered TA systems as well as refine the framework for recognizing the diverse biochemical functions in TA systems.

Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase.

Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.

A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis.

New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coliIMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism.

Mycobacterium tuberculosis (Mtb) encodes an exceptionally large number of toxin-antitoxin (TA) systems, supporting the hypothesis that TA systems are involved in pathogenesis. We characterized the putative Mtb Rv1044-Rv1045 TA locus structurally and functionally, demonstrating that it constitutes a bona fide TA system but adopts a previously unobserved antitoxicity mechanism involving phosphorylation of the toxin. While Rv1045 encodes the guanylyltransferase TglT functioning as a toxin, Rv1044 encodes the novel atypical serine protein kinase TakA, which specifically phosphorylates the cognate toxin at residue S78, thereby neutralizing its toxicity. In contrast to previous predictions, we found that Rv1044-Rv1045 does not belong to the type IV TA family because TglT and TakA interact with each other as substrate and kinase, suggesting an unusual type of TA system. Protein homology analysis suggests that other COG5340-DUF1814 protein pairs, two highly associated but uncharacterized protein families widespread in prokaryotes, might share this unusual antitoxicity mechanism.

The response regulator PhoP negatively regulates Yersinia pseudotuberculosis and Yersinia pestis biofilms.

A few Yersinia pseudotuberculosis strains form biofilms on the head of the nematode Caenorhabditis elegans, but numerous others do not. We show that a widely used Y. pseudotuberculosis strain, YPIII, is biofilm positive because of a mutation in phoP, which encodes the response regulator of a two-component system. For two wild-type Y. pseudotuberculosis that do not make biofilms on C. elegans, deletion of phoP was sufficient to produce robust biofilms. In Yersinia pestis, a phoP mutant made more extensive biofilms in vitro than did the wild type. Expression of HmsT, a diguanylate cyclase that positively regulates biofilms, is diminished in Y. pseudotuberculosis strains with functional PhoP.

Differential Gene Expression Patterns of Yersinia pestis and Yersinia pseudotuberculosis during Infection and Biofilm Formation in the Flea Digestive Tract.

Yersinia pestis, the etiologic agent of plague, emerged as a fleaborne pathogen only within the last 6,000 years. Just five simple genetic changes in the Yersinia pseudotuberculosis progenitor, which served to eliminate toxicity to fleas and to enhance survival and biofilm formation in the flea digestive tract, were key to the transition to the arthropodborne transmission route. To gain a deeper understanding of the genetic basis for the development of a transmissible biofilm infection in the flea foregut, we evaluated additional gene differences and performed in vivo transcriptional profiling of Y. pestis, a Y. pseudotuberculosis wild-type strain (unable to form biofilm in the flea foregut), and a Y. pseudotuberculosis mutant strain (able to produce foregut-blocking biofilm in fleas) recovered from fleas 1 day and 14 days after an infectious blood meal. Surprisingly, the Y. pseudotuberculosis mutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change the expression levels of the hms genes responsible for the synthesis and export of the extracellular polysaccharide matrix required for mature biofilm formation. The Y. pseudotuberculosis mutant uniquely expressed much higher levels of Yersinia type VI secretion system 4 (T6SS-4) in the flea, and this locus was required for flea blockage by Y. pseudotuberculosis but not for blockage by Y. pestis. Significant differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing-related genes, transcription regulation genes, and stress response genes were evident during flea infection. IMPORTANCE Y. pestis emerged as a highly virulent, arthropod-transmitted pathogen on the basis of relatively few and discrete genetic changes from Y. pseudotuberculosis. Parallel comparisons of the in vitro and in vivo transcriptomes of Y. pestis and two Y. pseudotuberculosis variants that produce a nontransmissible infection and a transmissible infection of the flea vector, respectively, provided insights into how Y. pestis has adapted to life in its flea vector and point to evolutionary changes in the regulation of metabolic and biofilm development pathways in these two closely related species.

AU-Rich Long 3' Untranslated Region Regulates Gene Expression in Bacteria.

3' untranslated regions (3' UTRs) and particularly long 3' UTRs have been shown to act as a new class of post-transcriptional regulatory element. We previously reported that hmsT mRNA stability is negatively regulated by the 3' UTR of hmsT in Yersinia pestis. To investigate more general effects of 3' UTRs in Y. pestis, we selected 15 genes potentially possessing long 3' UTRs with different AU content and constructed their 3' UTR deletion mutants. Deletion of AU-rich 3' UTRs increased mRNA levels, whereas deletion of 3' UTRs with normal AU content resulted in slight or no changes in the mRNA level. In addition, we found that PNPase was important for 3' UTR-mediated mRNA decay when the transcriptional terminator was Rho-dependent. Finally, we showed that ribosomes promote mRNA stability when bound to a 3' UTR. Our findings suggest that functional 3' UTRs might be broadly distributed in bacteria and their novel regulatory mechanisms require further investigation.

An acquired efflux system is responsible for copper resistance in Xanthomonas strain IG-8 isolated from China.

The genus Xanthomonas contains plant pathogens exhibiting innate resistance to a range of antimicrobial agents. In other genera, multidrug resistance is mediated by a synergy between a low-permeability outer membrane and expression of a number of multidrug efflux systems. This report describes the isolation of a novel gene cluster xmeRSA from Xanthomonas strain IG-8 that mediates copper chloride resistance. Subsequent analysis of these genes showed that they were responsible for the high level of multiple resistance in this strain and were homologues of the sme system of Stenotrophomonas maltophilia. Knock-out mutants of this gene cluster indicate that these genes are required for the copper resistance phenotype of strain IG-8. Expression analysis using lacZ fusions indicates that the genes are regulated by copper and other antimicrobials. Bioinformatic analysis suggests that these genes were acquired by horizontal gene transfer.

New Insights into the Non-orthodox Two Component Rcs Phosphorelay System.

The Rcs phosphorelay system, a non-orthodox two-component regulatory system, integrates environmental signals, regulates gene expression, and alters the physiological behavior of members of the Enterobacteriaceae family of Gram-negative bacteria. Recent studies of Rcs system focused on protein interactions, functions, and the evolution of Rcs system components and its auxiliary regulatory proteins. Herein we review the latest advances on the Rcs system proteins, and discuss the roles that the Rcs system plays in the environmental adaptation of various Enterobacteriaceae species.