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ABSTRACT Phylogenomic footprinting is an approach for ab initio identification of genome-wide regulatory elements in bacterial species based on sequence conservation. The statistical power of the phylogenomic approach depends on the degree of sequence conservation, the length of regulatory elements, and the level of phylogenetic divergence among genomes. Building on an earlier model, we propose a binomial model that uses synonymous tree lengths as neutral expectations for determining the statistical significance of conserved intergenic spacer (IGS) sequences. Simulations show that the binomial model is robust to variations in the value of evolutionary parameters, including base frequencies and the transition-to-transversion ratio. We used the model to search for regulatory sequences in the Lyme disease species group (Borrelia burgdorferi sensu lato) using 23 genomes. The model indicates that the currently available set of Borrelia genomes would not yield regulatory sequences shorter than five bases, suggesting that genome sequences of additional B. burgdorferi sensu lato species are needed. Nevertheless, we show that previously known regulatory elements are indeed strongly conserved in sequence or structure across these Borrelia species. Further, we predict with sufficient confidence two new RpoS binding sites, 39 promoters, 19 transcription terminators, 28 noncoding RNAs, and four sets of coregulated genes. These putative cis- and trans-regulatory elements suggest novel, Borrelia-specific mechanisms regulating the transition between the tick and host environments, a key adaptation and virulence mechanism of B. burgdorferi. Alignments of IGS sequences are available on BorreliaBase.org, an online database of orthologous open reading frame (ORF) and IGS sequences in Borrelia. IMPORTANCE While bacterial genomes contain mostly protein-coding genes, they also house DNA sequences regulating the expression of these genes. Gene regulatory sequences tend to be conserved during evolution. By sequencing and comparing related genomes, one can therefore identify regulatory sequences in bacteria based on sequence conservation. Here, we describe a statistical framework by which one may determine how many genomes need to be sequenced and at what level of evolutionary relatedness in order to achieve a high level of statistical significance. We applied the framework to Borrelia burgdorferi, the Lyme disease agent, and identified a large number of candidate regulatory sequences, many of which are known to be involved in regulating the phase transition between the tick vector and mammalian hosts.

Clostridioides difficile , the major cause of antibiotic-associated diarrhea, is a strict anaerobic, sporulating Firmicutes. However, during its infectious cycle, this anaerobe is exposed to low oxygen (O 2 ) tensions, with a longitudinal decreasing gradient along the gastrointestinal tract and a second lateral gradient with higher O 2 tensions in the vicinity of the cells. A plethora of enzymes involved in oxidative stress detoxication has been identified in C. difficile , including four O 2 -reducing enzymes: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). Here, we investigated the role of the four O 2 -reducing enzymes in the tolerance to increasing physiological O 2 tensions and air. The four enzymes have different, yet overlapping, spectra of activity. revRbr2 is specific to low O 2 tensions (<0.4%), FdpA to low and intermediate O 2 tensions (0.4%-1%), revRbr1 has a wider spectrum of activity (0.1%-4%), and finally FdpF is more specific to tensions > 4% and air. These different O 2 ranges of action partly arise from differences in regulation of expression of the genes encoding those enzymes. Indeed, we showed that revrbr2 is under the dual control of σ A and σ B . We also identified a regulator of the Spx family that plays a role in the induction of fdp and revrbr genes upon O 2 exposure. Finally, fdpF is regulated by Rex, a regulator sensing the NADH/NAD + ratio. Our results demonstrate that the multiplicity of O 2 -reducing enzymes of C. difficile is associated with different roles depending on the environmental conditions, stemming from a complex multi-leveled network of regulation., Importance: The gastrointestinal tract is a hypoxic environment, with the existence of two gradients of O 2 along the gut, one longitudinal anteroposterior decreasing gradient and one proximodistal increasing from the lumen to the epithelial cells. O 2 is a major source of stress for an obligate anaerobe such as the enteropathogen C. difficile . This bacterium possesses a plethora of enzymes capable of scavenging O 2 and reducing it to H 2 O. In this work, we identified the role of the four O 2 -reducing enzymes in the tolerance to the physiological O 2 tensions faced by C. difficile during its infectious cycle. These four enzymes have different spectra of action and protect the vegetative cells over a large range of O 2 tensions. These differences are associated with a distinct regulation of each gene encoding those enzymes. The complex network of regulation is crucial for C. difficile to adapt to the various O 2 tensions encountered during infection., Competing Interests: The authors declare no conflict of interest.

Cystic fibrosis (CF) airways are L-arginine deficient which may affect susceptibility to infection with certain pathogens. The potential impact of L-arginine supplementation on Pseudomonas aeruginosa , a common CF airway pathogen, is unclear. This study investigated the effects of L-arginine on P. aeruginosa biofilm cultures, using the laboratory strain PAO1 and multi-drug resistant CF clinical isolates. P. aeruginosa biofilms were grown in a chambered cover-glass slide model for 24 h, then exposed to either L-arginine alone or combined with tobramycin for an additional 24 h. Biofilms were visualized using confocal microscopy, and viable cells were measured via plating for colony-forming units. Increasing concentrations of L-arginine in bacterial culture medium reduced the biovolume of P. aeruginosa in a dose-dependent manner. Notably, L-arginine concentrations within the physiological range (50 mM and 100 mM) in combination with tobramycin promoted biofilm growth, while higher concentrations (600 mM and 1200 mM) inhibited growth. These findings demonstrate the potential of L-arginine as an adjuvant therapy to inhaled tobramycin in treating P. aeruginosa infections in people with CF., Competing Interests: The authors declare no conflict of interest.

Importance: During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti , the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission., Competing Interests: The authors declare no conflict of interest.

Agar dilution is the gold standard method for phenotypic antimicrobial susceptibility testing (AST) for Neisseria gonorrhoeae . However, this method is laborious and requires expertise, so laboratories that perform N. gonorrhoeae AST may choose alternative methods such as disk diffusion and gradient diffusion. In this study, we retrospectively compare the performance of gradient diffusion to agar dilution for 2,394 unique N. gonorrhoeae isolates identified in Alberta from 2017 to 2020 against azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, and tetracycline. Genome sequencing was utilized to resolve discrepancies between AST methods, detect antimicrobial resistance markers, and identify trends between error rates and sequence types (STs) of isolates. Over 90% of N. gonorrhoeae isolates were susceptible to azithromycin, cefixime, and ceftriaxone, whereas decreased susceptibility was observed for ciprofloxacin, penicillin, and tetracycline. Categorical (CA) and essential agreement (EA) was poorest between the two methods for penicillin (CA: 86.02%; EA: 77.69%) and tetracycline (CA: 47.22%; EA: 55.96%); however, the low CA was primarily attributed to minor errors. Antimicrobial agents with errors outside of acceptable limits included azithromycin (very major error: 18.42%; major error: 7.73%) and tetracycline (very major error: 6.17%). Genome sequencing on a subset of isolates resolved 30.3% of the azithromycin major errors and confirmed the azithromycin or tetracycline very major errors. Significant associations between certain STs and error types for azithromycin and tetracycline were also identified. Overall, gradient diffusion compared well to agar dilution for cefixime, ceftriaxone, and ciprofloxacin, and genome sequencing was identified as a useful tool to arbitrate discrepant susceptibility testing results between gradient diffusion and agar dilution for N. gonorrhoeae ., Competing Interests: Tanis C. Dingle is a voting member on the CLSI Antimicrobial Susceptibility Testing subcommittee. Tanis C. Dingle has received speaker honoraria from BioMerieux.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way., Competing Interests: The authors declare no conflict of interest.

We present the draft genome of a Wolbachia endosymbiont from quill mites. This is the first representative of a recently discovered distinct Wolbachia lineage (supergroup P). We hope the genome will be a useful resource for comparative evolutionary and genomic studies across the globally distributed symbiont Wolbachia ., Competing Interests: The authors declare no conflict of interest.

Obligate anaerobic bacteria in genus Faecalibacterium are among the most dominant taxa in the colon of healthy individuals and contribute to intestinal homeostasis. A decline in the abundance of this genus is associated with the occurrence of various gastrointestinal disorders, including inflammatory bowel diseases. In the colon, these diseases are accompanied by an imbalance between the generation and elimination of reactive oxygen species (ROS), and oxidative stress is closely linked to disruptions in anaerobiosis. In this work, we explored the impact of oxidative stress on several strains of faecalibacteria. An in silico analysis of complete genomes of faecalibacteria revealed the presence of genes encoding O 2 - and/or ROS-detoxifying enzymes, including flavodiiron proteins, rubrerythrins, reverse rubrerythrins, superoxide reductases, and alkyl peroxidase. However, the presence and the number of these detoxification systems varied greatly among faecalibacteria. These results were confirmed by O 2 stress survival tests, in which we found that strains differed widely in their sensitivity. We showed the protective role of cysteine, which limited the production of extracellular O 2 •- and improved the survival of Faecalibacterium longum L2-6 under high O 2 tension. In the strain F. longum L2-6, we observed that the expression of genes encoding detoxifying enzymes was upregulated in the response to O 2 or H 2 O 2 stress but with different patterns of regulation. Based on these results, we propose a first model of the gene regulatory network involved in the response to oxidative stress in F. longum L2-6. IMPORTANCE Commensal bacteria in the genus Faecalibacterium have been proposed for use as next-generation probiotics, but efforts to cultivate and exploit the potential of these strains have been limited by their sensitivity to O 2 . More broadly, little is known about how commensal and health-associated bacterial species in the human microbiome respond to the oxidative stress that occurs as a result of inflammation in the colon. In this work, we provide insights regarding the genes that encode potential mechanisms of protection against O 2 or ROS stress in faecalibacteria, which may facilitate future advances in work with these important bacteria., Competing Interests: The authors declare no conflict of interest.

With improvement in laboratory diagnosis of Mycoplasmoides genitalium infection through molecular diagnostics, macrolide resistance determination within M. genitalium-positive patients is necessary. In this study, we report baseline parameters for an analyte-specific reagent (ASR) macrolide resistance real-time reverse transcriptase PCR on an open access analyzer and evaluated detection of macrolide resistance-mediated mutation (MRM) within 23S rRNA in a clinical specimen set. Initial use of 1.2 μM M. genitalium primer and 0.8 μM M. genitalium detection probe concentrations yielded an 80% false-positive detection rate when challenged with 10,000 copies of wild-type RNA. Optimization experiments showed that lowering primer/detection probe and MgCl 2 concentrations minimized these false-detections of wild-type 23S rRNA, while higher levels of KCl increased rates of MRM detection with concomitant lower cycle threshold values and higher fluorescence emission. Lower limit of A2058G mutation detection was 5000 copies/mL (180 copies/reaction; 20/20 detections). Utilization of a baseline correction slope limit of 250 units further mitigated false-detection from wild-type 23S rRNA at challenges up to 3.3 billion copies/mL. MRM was detected in 583/866 (67.3%) clinical specimens initially positive for M. genitalium by commercial transcription-mediated amplification. These data included 392/564 detections (69.5%) from M. genitalium-positive swab specimens and 191/302 (63.2%) from M. genitalium-positive-positive first-void urine specimens ( P =  0.06). Overall resistance detection rates did not vary by gender ( P =  0.76). Specificity of the M. genitalium macrolide resistance ASR was 100% (141 urogenital determinations). MRM detection by the ASR was confirmed at a concordance rate of 90.9% by Sanger sequencing of a clinical specimen subset., Competing Interests: The authors declare a conflict of interest. E.M. has received honoraria and travel support from Hologic, Incorporated.

Gonorrhea is a sexually transmitted bacterial infection caused by Neisseria gonorrhoeae. Nucleic acid amplification testing is the preferred method for routine diagnosis of gonorrhea from urogenital specimens, but culture is commonly used for diagnosis of disseminated infections, including gonococcal arthritis. The Hologic Aptima Combo 2 (AC2), a transcription-mediated amplification assay, is FDA and Health Canada licensed for detection of N. gonorrhoeae and Chlamydia trachomatis from urogenital, rectal, and pharyngeal specimens, but not joint fluid. In the current study, we compared the performance of microscopy, culture, and the AC2 for detection of N. gonorrhoeae from 170 joint fluid specimens. A total of five specimens were culture-positive, whereas 14 were AC2-positive. Gram-negative diplococci, characteristic of Neisseria , were observed in only two joint fluid specimens. Complementary testing confirmed the presence of N. gonorrhoeae in seven discordant (i.e., culture-negative/AC2-positive) specimens. These results indicate that the AC2 is more sensitive than culture for the diagnosis of gonococcal arthritis.

Azithromycin-resistant (AZIR) gonorrhea has been steadily increasing in Canada over the past decade, which is cause for alarm, as azithromycin (AZI) has been part of the combination therapy recommended by the Canadian Guidelines on Sexually Transmitted Infections (CGSTI) since 2012. Neisseria gonorrhoeae with AZI MICs ≥1 mg/L collected between 2015 and 2018 as part of the Gonococcal Antimicrobial Surveillance Program-Canada underwent antimicrobial susceptibility testing, molecular typing, and whole-genome sequencing. Regional, demographic, and clinical isolation site comparisons were made to aid in our understanding of AZI susceptibility trending. We identified 3,447 N. gonorrhoeae with AZI MICs of ≥1 mg/L in Canada, which increased from 6.3% in 2015 to 26.5% of isolates in 2018. Central Canada had the highest proportion, rising from 9.2% in 2015 to 31.2% in 2018. We identified 273 different N. gonorrhoeae multiantigen sequence types (NG-MAST) among these isolates, with ST-12302 the most prevalent (50.9%). Whole-genome sequencing identified the Neisseria lactamica-like mosaic mtr locus as the mechanism of AZIR in isolates of ST-12302 and isolates genetically similar (differing by ≤5 bp), designated the ST-12302 genogroup, accounting for 65.2% of study isolates which were originally identified in central Canada but spread to other regions by 2018. Genomic analysis indicated that AZIR in Canadian N. gonorrhoeae expanded rapidly due to clonal spread of the ST-12302 genogroup. The rapid expansion of this AZIR clonal group in all regions of Canada is of concern. CGSTI are currently under review to address the increase in AZIR in Canada.

Antimicrobial resistance in Streptococcus pneumoniae represents a threat to public health, and monitoring the dissemination of resistant strains is essential to guiding health policy. Multiple-variable linear regression modeling was used to determine the contributions of molecular antimicrobial resistance determinants to antimicrobial MICs for penicillin, ceftriaxone, erythromycin, clarithromycin, clindamycin, levofloxacin, and trimethoprim-sulfamethoxazole. Training data sets consisting of Canadian S. pneumoniae isolates obtained from 1995 to 2019 were used to generate multiple-variable linear regression equations for each antimicrobial. The regression equations were then applied to validation data sets of Canadian ( n  = 439) and U.S. ( n  = 607 and n  = 747) isolates. The MICs for β-lactam antimicrobials were fully explained by amino acid substitutions in motif regions of the penicillin binding proteins PBP1a, PPB2b, and PBP2x. Accuracies of predicted MICs within 1 doubling dilution to phenotypically determined MICs were 97.4% for penicillin, 98.2% for ceftriaxone, 94.8% for erythromycin, 96.6% for clarithromycin, 98.2% for clindamycin, 100% for levofloxacin, and 98.8% for trimethoprim-sulfamethoxazole, with an overall sensitivity of 95.8% and specificity of 98.0%. Accuracies of predicted MICs to the phenotypically determined MICs were similar to those of phenotype-only MIC comparison studies. The ability to acquire detailed antimicrobial resistance information directly from molecular determinants will facilitate the transition from routine phenotypic testing to whole-genome sequencing analysis and can fill the surveillance gap in an era of increased reliance on nucleic acid assay diagnostics to better monitor the dynamics of S. pneumoniae.

Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division. IMPORTANCE Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites. How these enzymes are regulated remains poorly understood. We identify a new PG hydrolase involved in cell division, CwlA, in the enteropathogen C. difficile Lack or accumulation of CwlA at the bacterial surface is responsible for a division defect, while its accumulation in the absence of PrkC also increases susceptibility to antimicrobial compounds targeting the cell wall. CwlA is a substrate of the kinase PrkC in C. difficile PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, consequently, its activity in the cell wall. This work provides a novel regulatory mechanism by STK in tightly controlling protein export., (Copyright © 2021 Garcia-Garcia et al.)

Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O 2 ) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O 2 ) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O 2 detoxification and/or protection. Tolerance of C. difficile to low O 2 tensions requires the presence of the alternative sigma factor, σ B , involved in the general stress response. Among the genes positively controlled by σ B , four encode proteins likely involved in O 2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O 2 - and H 2 O 2 -reductase activities in vitro , while purified FdpA mainly acts as an O 2 -reductase. The growth of a fdpA mutant is affected at 0.4% O 2 , while inactivation of both revRbrs leads to a growth defect above 0.1% O 2 O 2 -reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O 2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O 2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O 2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O 2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O 2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile This enteropathogen has developed efficient strategies to detoxify O 2 In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O 2 tolerance in C. difficile These enzymes are responsible for the reduction of O 2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O 2 -reductases are crucial in the ability of C. difficile to tolerate O 2 and survive to O 2 concentrations encountered in the gastrointestinal tract., (Copyright © 2020 Kint et al.)

Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para -cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para -hydroxyphenylacetate ( p -HPA) to produce p -cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA , and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p -HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P 1 , P 2 , and Pσ 54 ; only the P 1 promoter was responsible for both basal and p -HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p -HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p -HPA in minimal media. We show that induction of the hpdBCA operon in response to p -HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N 13 -CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p -HPA, which drives the production of p -cresol. This provides insights into the regulatory control of p -cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis. IMPORTANCE Clostridioides difficile infection results from antibiotic-associated dysbiosis. para -Cresol, a phenolic compound produced by C. difficile , selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p -HPA to p -cresol, is upregulated in response to elevated exogenous p -HPA, with induction occurring between >0.1 and ≤0.25 mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p -HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p -HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p -HPA was required for p -cresol production. Identifying regulatory controls of p -cresol production will provide novel therapeutic targets to prevent p -cresol production, reducing C. difficile 's competitive advantage., (© Crown copyright 2020.)

Whole-genome sequencing was used to identify mutations in antibiotic resistance-conferring genes to compare susceptibility predictions with MICs and to ascertain strain types in 99 isolates of Neisseria gonorrhoeae Genotypes associated with susceptibility, as well as MIC creep or emerging resistance, were noted. Phylogenomic analysis revealed three distinctive clades and putative gonococcal transmission linkages involving a tetracycline-resistant N. gonorrhoeae outbreak and the clonal spread of susceptible isolates in men., (Copyright © 2020 American Society for Microbiology.)

The emergence of Neisseria gonorrhoeae strains that are resistant to azithromycin and extended-spectrum cephalosporins represents a public health threat, that of untreatable gonorrhea infections. Multivariate regression modeling was used to determine the contributions of molecular antimicrobial resistance determinants to the overall antimicrobial MICs for ceftriaxone, cefixime, azithromycin, tetracycline, ciprofloxacin, and penicillin. A training data set consisting of 1,280 N. gonorrhoeae strains was used to generate regression equations which were then applied to validation data sets of Canadian ( n  = 1,095) and international ( n  = 431) strains. The predicted MICs for extended-spectrum cephalosporins (ceftriaxone and cefixime) were fully explained by 5 amino acid substitutions in PenA, A311V, A501P/T/V, N513Y, A517G, and G543S; the presence of a disrupted mtrR promoter; and the PorB G120 and PonA L421P mutations. The correlation of predicted MICs within one doubling dilution to phenotypically determined MICs of the Canadian validation data set was 95.0% for ceftriaxone, 95.6% for cefixime, 91.4% for azithromycin, 98.2% for tetracycline, 90.4% for ciprofloxacin, and 92.3% for penicillin, with an overall sensitivity of 99.9% and specificity of 97.1%. The correlations of predicted MIC values to the phenotypically determined MICs were similar to those from phenotype MIC-only comparison studies. The ability to acquire detailed antimicrobial resistance information directly from molecular data will facilitate the transition to whole-genome sequencing analysis from phenotypic testing and can fill the surveillance gap in an era of increased reliance on nucleic acid assay testing (NAAT) diagnostics to better monitor the dynamics of N. gonorrhoeae ., (© Crown copyright 2020.)

Clostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A Δ prkC mutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a Δ prkC mutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the Δ prkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the Δ prkC mutant than the wild-type strain. Finally, in a hamster model of infection, the Δ prkC mutant had a colonization delay that did not significantly affect overall virulence., (Copyright © 2019 American Society for Microbiology.)

A real-time PCR (RT-PCR) assay was designed for the simultaneous identification of Neisseria gonorrhoeae and its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) in gyrA of N. gonorrhoeae The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254 N. gonorrhoeae isolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non- N. gonorrhoeae species isolates. The performance of the primers was validated using genomic DNA from 100 different N. gonorrhoeae isolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non- N. gonorrhoeae isolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of the N. gonorrhoeae isolates and 23 non- N. gonorrhoeae isolates. These primers detected N. gonorrhoeae and its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status., (Copyright © 2017 American Society for Microbiology.)

A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible (https://ngstar.canada.ca). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes ( penA , mtrR , porB , ponA , gyrA , parC , and 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA , porB , gyrA , and parC ; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) ( n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs ( n = 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 ( n = 100; 13.0%), ST-42 and ST-91 ( n = 45; 5.9%), ST-64 ( n = 44; 5.72%), and ST-139 ( n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 ( n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 ( n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 ( n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 ( n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains., (© Crown copyright 2017.)

Using serotyping, multilocus sequence typing, and whole-genome sequencing (WGS) of selected strains, we studied the population structure of 102 group B Streptococcus (GBS) isolates prospectively sampled in 2014 from vaginal/rectal swabs of healthy pregnant women in metropolitan Toronto, Canada. We also determined the susceptibilities of each of the colonizing isolates to penicillin, erythromycin, clindamycin, tetracycline, and other antimicrobial agents. Overall, we observed a high rate of tetracycline resistance (89%) among colonizing GBS isolates. We found resistance to erythromycin in 36% of the strains, and 33% were constitutively or inducibly resistant to clindamycin. The most frequently identified serotypes were III (25%), Ia (23%), and V (19%). Serotype IV accounted for 6% of the colonizing isolates, a rate consistent with that observed among patients with invasive GBS infections in metropolitan Toronto. The majority of serotype IV isolates belonged to sequence type (ST)459, a tetracycline-, erythromycin-, and clindamycin-resistant ST first identified in Minnesota, which is considered to be the main driver of serotype IV GBS expansion in North America. WGS revealed that ST459 isolates from Canada are clonally related to colonizing and invasive ST459 organisms circulating in regions of the United States. We also used WGS to study recombination in selected colonizing strains from metropolitan Toronto, which revealed multiple episodes of capsular switching. Present and future circulating GBS organisms and their genetic diversity may influence GBS vaccine development., (Copyright © 2017 American Society for Microbiology.)

Neisseria gonorrhoeae strains with coresistance to the first-line antimicrobial treatments azithromycin and ceftriaxone are an emerging public health threat. Here, we present the complete genome sequences of three strains of N. gonorrhoeae, including one susceptible strain and two strains with coresistance to ceftriaxone and azithromycin., (© Crown copyright 2016.)

The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Rectal swabs from high-risk patients were screened for carbapenem-resistant organisms (CROs) using several methods. The direct MacConkey plate method was the most sensitive for CROs (95%), while chromID CARBA and the Check-Direct CPE screen assay were the most sensitive for the detection of carbapenemase-producing organisms (CPOs) (100%; all blaKPC). All methods had a specificity of >90% for CROs, and for CPOs, the specificity ranged from 85 to 98%. Broth enrichment methods performed poorly compared to direct inoculation methods, negating the need for the broth enrichment step., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZM(r)) represents a public health threat of untreatable gonorrhea infections. Genomic epidemiology through whole-genome sequencing was used to describe the emergence, dissemination, and spread of AZM(r) strains. The genomes of 213 AZM(r) and 23 AZM-susceptible N. gonorrhoeae isolates collected in Canada from 1989 to 2014 were sequenced. Core single nucleotide polymorphism (SNP) phylogenomic analysis resolved 246 isolates into 13 lineages. High-level AZM(r) (MICs ≥ 256 μg/ml) was found in 5 phylogenetically diverse isolates, all of which possessed the A2059G mutation (Escherichia coli numbering) in all four 23S rRNA alleles. One isolate with high-level AZM(r) collected in 2009 concurrently had decreased susceptibility to ceftriaxone (MIC = 0.125 μg/ml). An increase in the number of 23S rRNA alleles with the C2611T mutations (E. coli numbering) conferred low to moderate levels of AZM(r) (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZM(r) was also associated with mtrR promoter mutations, including the -35A deletion and the presence of Neisseria meningitidis-like sequences. Geographic and temporal phylogenetic clustering indicates that emergent AZM(r) strains arise independently and can then rapidly expand clonally in a region through local sexual networks., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Data from the Canadian National Gonococcal Antimicrobial Susceptibility Comparison Program, including results from 25 proficiency panels distributed between 2003 and 2012, were analyzed. The average MIC agreement between the participating laboratories ranged from 85.6% to 98.8% over the 10-year period, with the interpretation agreement ranging from 85.7% to 98.1%., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Serotype IV group B Streptococcus (GBS) is emerging in Canada and the United States with rates as high as 5% of the total burden of adult invasive GBS disease. To understand this emergence, we studied the population structure and assessed the antimicrobial susceptibility of serotype IV isolates causing adult invasive infection in Manitoba and Saskatchewan, Canada, between 2010 and 2014. Whole-genome sequencing was used to determine multilocus sequence typing information and identify genes encoding antimicrobial resistance in 85 invasive serotype IV GBS strains. Antimicrobial susceptibility testing was performed by standard methods. Strain divergence was assessed using genome-wide single-nucleotide polymorphism analysis. Serotype IV strains were responsible for 16.9% of adult invasive GBS infections in Manitoba and Saskatchewan during the period. The majority of serotype IV isolates (89%) were clonally related, tetracycline-, erythromycin-, and clindamycin-resistant sequence type 459 (ST459) strains that possessed genes tetM and ermTR. Genome comparisons between ST459 and serotype V ST1 GBS identified several areas of recombination in an overall similar genomic background. Serotype IV ST459 GBS strains are expanding and causing a substantial percentage of adult invasive GBS disease. This emergence may be linked to the acquisition of resistance to tetracycline, macrolides, and lincosamides., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

To characterize the relationship between penicillin-binding protein 2 (PBP2/penA) and susceptibility to extended-spectrum cephalosporins (ESCs) and carbapenem antibiotics, we compared 17 PBP2 variants in Neisseria gonorrhoeae. Nonmosaic and mosaic variants of PBP2 caused decreased susceptibility to ESCs and, to a lesser extent, to carbapenems. An A501P substitution in mosaic XXXIV&#95;A501P conferred decreased susceptibility to ESCs but restored carbapenem susceptibility to wild-type levels. These results could aid the molecular surveillance of antimicrobial resistance to these agents., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A large-scale, whole-genome comparison of Canadian Neisseria gonorrhoeae isolates with high-level cephalosporin MICs was used to demonstrate a genomic epidemiology approach to investigate strain relatedness and dynamics. Although current typing methods have been very successful in tracing short-chain transmission of gonorrheal disease, investigating the temporal evolutionary relationships and geographical dissemination of highly clonal lineages requires enhanced resolution only available through whole-genome sequencing (WGS). Phylogenomic cluster analysis grouped 169 Canadian strains into 12 distinct clades. While some N. gonorrhoeae multiantigen sequence types (NG-MAST) agreed with specific phylogenomic clades or subclades, other sequence types (ST) and closely related groups of ST were widely distributed among clades. Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) emerged among a group of diverse strains in Canada during the 1990s with a variety of nonmosaic penA alleles, followed in 2000/2001 with the penA mosaic X allele and then in 2007 with ST1407 strains with the penA mosaic XXXIV allele. Five genetically distinct ESC-DS lineages were associated with penA mosaic X, XXXV, and XXXIV alleles and nonmosaic XII and XIII alleles. ESC-DS with coresistance to azithromycin was observed in 5 strains with 23S rRNA C2599T or A2143G mutations. As the costs associated with WGS decline and analysis tools are streamlined, WGS can provide a more thorough understanding of strain dynamics, facilitate epidemiological studies to better resolve social networks, and improve surveillance to optimize treatment for gonorrheal infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Azithromycin (AZM) is routinely recommended as a component of dual therapy for gonorrhea in combination with third-generation cephalosporins (3GC). In this study, we examined the prevalence of AZM-resistant (AZM(r)) Neisseria gonorrhoeae from July 2010 to February 2013, assessed the rate of concurrent cephalosporin resistance under the current treatment recommendations, and analyzed the clonal distribution of AZM(r) isolates in Ontario, Canada. Nineteen AZM(r) clinical isolates (one per patient; MIC, ≥2 μg/ml) were included in the study. Susceptibility profiles of these isolates to 11 antibiotics, molecular typing, characterization of macrolide resistance mechanisms, and penicillin-binding protein 2 (PBP2) patterns were determined for all the isolates. Two groups were defined based on AZM(r) level; group A isolates displayed high-level resistance (MIC, ≥2,048 μg/ml) due to mutations (A2143G) in the four copies of the 23S rRNA rrl gene, and group B isolates had moderate resistance to AZM (MICs, 2 to 8 μg/ml, C2599T mutation in the rrl gene), with a subgroup belonging to sequence type 3158 (ST3158) (n = 8), which also showed reduced susceptibility to 3GC (MICs, 0.12 to 0.25 μg/ml, PBP2 pattern XXXIV). This AZM(r) phenotype was not observed in previous provincial surveillance in 2008 (the ST3158 clone was found, with AZM MICs of 0.25 to 0.5 μg/ml associated with mtrR mutations). We hypothesized that the AZM mutant prevention concentration (MPC) in the ST3158 subpopulation we found in 2008 was higher than the MPC in wild-type isolates (AZM MIC, ≤0.031 μg/ml), increasing the chances of additional selection of AZM(r) mutations. Full AZM resistance is now emerging in this clone together with reduced susceptibility to 3GC, threatening the future efficacy of these antibiotics as therapeutic options for treatment of gonorrhea.

We determined the capsular polysaccharide (CPS) type of 600 group B Streptococcus (GBS) (also known as Streptococcus agalactiae) strains recovered from patients with invasive infections in the greater Toronto area, Canada, between 2009 and 2012. GBS strains of CPS type III were the most prevalent among infants (44% in those with early-onset disease, 75% in those with late-onset disease), while type V strains were most frequently isolated from adult patients (26% in patients≥19 years old). We next investigated the presence in our collection of GBS strains belonging to the hypervirulent multilocus sequence typing clonal complex 17 (CC17). We used a PCR test described as specific for the detection of CC17 strains, which targets the gene encoding the major virulence factor HvgA. We identified 91 hvgA-positive strains; of these, 88 were CPS type III, 2 were CPS type IV, and 1 was CPS type V. Using whole-genome sequencing, we showed that the two hvgA-positive CPS type IV strains are CC17 strains which underwent capsular switching. However, sequence analysis revealed that the hvgA-positive CPS type V strain does not belong to CC17 but instead is a bona fide CC1 strain which acquired hvgA, probably by recombination from a CC17 donor. Our findings underline the importance of recombination in GBS pathogenesis and caution against the use of single-gene-based PCR tests to detect CC17 GBS strains.

We evaluated antimicrobial resistance in Neisseria gonorrhoeae isolated from men enrolled in a randomized trial of male circumcision to prevent HIV. Urethral specimens from men with discharge were cultured for N. gonorrhoeae. MICs were determined by agar dilution. Clinical and Laboratory Standards Institute (CLSI) criteria defined resistance: penicillin, tetracycline, and azithromycin MICs of ≥2.0 μg/ml; a ciprofloxacin MIC of ≥1.0 μg/ml; and a spectinomycin MIC of ≥128.0 μg/ml. Susceptibility to ceftriaxone and cefixime was shown by an MIC of ≤0.25 μg/ml. Additionally, PCR amplification identified mutations in parC and gyrA genes in selected isolates. From 2002 to 2009, 168 N. gonorrhoeae isolates were obtained from 142 men. Plasmid-mediated penicillin resistance was found in 65%, plasmid-mediated tetracycline resistance in 97%, and 11% were ciprofloxacin resistant (quinolone-resistant N. gonorrhoeae [QRNG]). QRNG appeared in November 2007, increasing from 9.5% in 2007 to 50% in 2009. Resistance was not detected for spectinomycin, cefixime, ceftriaxone, or azithromycin, but MICs of cefixime (P = 0.018), ceftriaxone (P < 0.001), and azithromycin (P = 0.097) increased over time. In a random sample of 51 men, gentamicin MICs were as follows: 4 μg/ml (n = 1), 8 μg/ml (n = 49), and 16 μg/ml (n = 1). QRNG increased rapidly and alternative regimens are required for N. gonorrhoeae treatment in this area. Amid emerging multidrug-resistant N. gonorrhoeae, antimicrobial resistance surveillance is essential for effective drug choice. High levels of plasmid-mediated resistance and increasing MICs for cephalosporins suggest that selective pressure from antibiotic use is a strong driver of resistance emergence.

Toxin synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis. Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile. SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB, encoding the toxins, and of tcdR, encoding the sigma factor required for toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets.

In this study, we combined metabolic reconstruction, growth assays, and metabolome and transcriptome analyses to obtain a global view of the sulfur metabolic network and of the response to sulfur availability in Brevibacterium aurantiacum. In agreement with the growth of B. aurantiacum in the presence of sulfate and cystine, the metabolic reconstruction showed the presence of a sulfate assimilation pathway, thiolation pathways that produce cysteine (cysE and cysK) or homocysteine (metX and metY) from sulfide, at least one gene of the transsulfuration pathway (aecD), and genes encoding three MetE-type methionine synthases. We also compared the expression profiles of B. aurantiacum ATCC 9175 during sulfur starvation or in the presence of sulfate. Under sulfur starvation, 690 genes, including 21 genes involved in sulfur metabolism and 29 genes encoding amino acids and peptide transporters, were differentially expressed. We also investigated changes in pools of sulfur-containing metabolites and in expression profiles after growth in the presence of sulfate, cystine, or methionine plus cystine. The expression of genes involved in sulfate assimilation and cysteine synthesis was repressed in the presence of cystine, whereas the expression of metX, metY, metE1, metE2, and BL613, encoding a probable cystathionine-γ-synthase, decreased in the presence of methionine. We identified three ABC transporters: two operons encoding transporters were transcribed more strongly during cysteine limitation, and one was transcribed more strongly during methionine depletion. Finally, the expression of genes encoding a methionine γ-lyase (BL929) and a methionine transporter (metPS) was induced in the presence of methionine in conjunction with a significant increase in volatile sulfur compound production.

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.

MetR (formerly Smu.1225), a regulator of the LysR family, controls key genes for methionine supply in Streptococcus mutans. An S. mutans metR mutant is unable to transport l-methionine and to grow in the absence of this amino acid. Accordingly, MetR activates transcription by binding to the promoter regions of two gene clusters and smu.1487, whose products are involved in methionine biosynthesis (MetEF and Smu.1487) and uptake (AtmBDE). Transcriptional activation by MetR requires the presence of a 17-bp palindromic sequence, the Met box. Base substitutions in the Met box hinder the formation of a MetR-DNA complex and abolish MetR-dependent activation, showing that Met boxes correspond to MetR recognition sites. Activation by MetR occurs in methionine-depleted medium and is rapidly triggered under nonactivating conditions by the addition of homocysteine. This intermediate of methionine biosynthesis increases the affinity of MetR for DNA in vitro and appears to be the MetR coeffector in vivo. Homocysteine plays a crucial role in methionine metabolic gene regulation by controlling MetR activity. A similar mechanism of homocysteine- and MetR-dependent control of methionine biosynthetic genes operates in S. thermophilus. These data suggest a common mechanism for the regulation of the methionine supply in streptococci. However, some streptococcal species are unable to synthesize the homocysteine coeffector. This intriguing feature is discussed in the light of comparative genomics and streptococcal ecology.

Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine beta-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine gamma-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an DeltayrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a DeltayrhA DeltacysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.