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Staphylococcus aureus, as well as coagulase-negative staphylococci (CoNS), can cause a wide range of human infections both in nosocomial and community settings. Βeta-lactams are the antibiotics of choice for the treatment of bloodstream infections (BSI) caused by these microorganisms. Resistance to virtually all β-lactams (also referred to as methicillin resistance) primarily results from the production of an alternative penicillin-binding protein (PBP2a) encoded by the mecA gene. While β-lactams are still used as first-line therapy against BSI caused by S. aureus, BSI with CoNS are usually treated with vancomycin due to the high prevalence of methicillin resistance. Rapid detection of methicillin resistance is thus critical for continuation or adjustment of the empirical therapy and therewith to improve the clinical outcome of the patients. The revised version of the immunochromatographic assay PBP2a SA culture colony test (SACCT) is a rapid, inexpensive, and easy method that enables reliable detection of PBP2a in mecA-positive staphylococcal isolates after18 to 24 h of incubation. Here, we evaluated the diagnostic performance of the SACCT using primary subcultures of spiked blood cultures after short incubation (4 to 6 h) and established a modified procedure with an equal analytical performance to that of longer-grown cultures. With the proposed method the SACCT can be employed for PBP2a detection from shortly incubated subcultures of clinically relevant staphylococcal isolates, thereby allowing more rapid and effective management of BSI caused by these organisms. IMPORTANCE Antibiotic resistance poses a major threat to health and incurs high economic costs worldwide. Rapid detection of resistance mechanisms can contribute to improving patient care and preventing the dissemination of antimicrobial resistance. Here, we describe a rapid method to detect the most important beta-lactam resistance mechanism (the plasmid-encoded alternative transpeptidase PBP2a) in staphylococcal isolates causing BSI. We show that, using a modified procedure, PBP2a can be reliably detected from primary subcultures of spiked blood cultures after short incubation (4 to 6 h) with a rapid, inexpensive, and simple immunochromatographic test (SACCT). We provide an accurate, inexpensive, and rapid method to facilitate appropriate management and control of infections in patients suffering from invasive staphylococcal infections.

Nocardiosis is known to be an opportunistic infection most commonly affecting immunocompromised patients that can lead to life-threatening conditions. Primary cutaneous disease remains a rare manifestation and unlike pulmonary or disseminated nocardiosis, it usually affects immunocompetent individuals. We present a case of a primary cutaneous nocardiosis of the head and neck after an insect bite in a healthy 50-year-old woman who had recently travelled from Greece. She presented with a painful right-sided swelling of her face and neck and an ulcerated plaque over the right temple. Biopsy of the plaque revealed inflammation with abscess formation indicating underlying infection. Culture from the biopsy showed growth ofNocardiaspp and 16S rRNA gene sequence analysis identifiedNocardia brasiliensis. The patient was treated with trimethoprim/sulfamethoxazole and subsequently switched to amoxicillin/clavulanic acid due to a drug eruption. Antibiotic therapy was continued for a total of 3 months with complete resolution of the skin lesions.

Whole genome sequencing (WGS) enables high resolution typing of bacteria up to the single nucleotide polymorphism (SNP) level. WGS is used in clinical microbiology laboratories for infection control, molecular surveillance and outbreak analyses. Given the large palette of WGS reagents and bioinformatics tools, the Swiss clinical bacteriology community decided to conduct a ring trial (RT) to foster harmonization of NGS-based bacterial typing. The RT aimed at assessing methicillin-susceptible Staphylococcus aureus strain relatedness from WGS and epidemiological data. The RT was designed to disentangle the variability arising from differences in sample preparation, SNP calling and phylogenetic methods. Nine laboratories participated. The resulting phylogenetic tree and cluster identification were highly reproducible across the laboratories. Cluster interpretation was, however, more laboratory dependent, suggesting that an increased sharing of expertise across laboratories would contribute to further harmonization of practices. More detailed bioinformatic analyses unveiled that while similar clusters were found across laboratories, these were actually based on different sets of SNPs, differentially retained after sample preparation and SNP calling procedures. Despite this, the observed number of SNP differences between pairs of strains, an important criterion to determine strain relatedness given epidemiological information, was similar across pipelines for closely related strains when restricting SNP calls to a common core genome defined by S. aureus cgMLST schema. The lessons learned from this pilot study will serve the implementation of larger-scale RT, as a mean to have regular external quality assessments for laboratories performing WGS analyses in a clinical setting., Frontiers in Microbiology, 11, ISSN:1664-302X

SUMMARY Objective The international outbreak of cardiac-surgery-associated Mycobacterium chimaera infections was traced back to infectious aerosols originating from contaminated water reservoirs of heater-cooler devices (HCDs). In general, non-tuberculous mycobacteria (NTM) frequently colonize water systems and can contaminate medical devices. Data on detection of NTM other than M. chimaera in samples gathered from HCDs are scarce. The present study summarizes prospective mycobacterial surveillance of five HCDs over more than 4 years. Methods Five LivaNova 3T (London, UK) HCDs, acquired factory-new in 2014, were followed prospectively. Until mid-April 2014, the HCDs were maintained according to the manufacturer's recommendations, and subsequently, they were maintained according to an intensified in-house protocol including exhaust air evacuation. Mycobacterial surveillance cultures consisted of monthly water samples gathered from patient and cardioplegia circuits, as well as airflow samples. Results Of 441 water samples, 170 (38.6%) revealed NTM growth. The most frequently detected NTM were M. chimaera [N=120 (67.4%)], Mycobacterium gordonae [N=35 (19.7 %)] and Mycobacterium paragordonae [N=17 (9.6%)]. Growth of NTM, M. chimaera and M. paragordonae was significantly more common in water samples derived from the patient circuit than the cardioplegia circuit of the HCD. Three (2.0%) of 150 air samples grew NTM. Conclusion Growth of NTM in HCD water samples was common. Diverse NTM species were detected, with M. chimaera being the most common. The majority of air samples remained negative. The relevance of NTM other than M. chimaera contaminating HCDs is poorly defined, but a recent report on an HCD-associated outbreak with Mycobacterium abscessus confirmed a potential threat.

The co-occurrence of aminoglycoside and β-lactam resistance was assessed in 3358 consecutive Escherichia coli clinical isolates collected in 2014 in the greater Zurich area, Switzerland. Non-susceptibility to at least one of the tested aminoglycosides was observed in 470/3358 E. coli strains (14%). In strains categorized as broad-spectrum β-lactamase (BSBL)-producers (1241/3358 isolates), extended-spectrum β-lactamase (ESBL)-producers (262/3358) and AmpC-producers (66/3358), resistance to aminoglycoside was found in 23%, 52% and 20% of the isolates, respectively. In contrast, aminoglycoside-susceptible strains were rarely resistant to β-lactams (33/1777, 1.9%). The genomes of 439 aminoglycoside-resistant E. coli were sequenced and aminoglycoside and β-lactam genotypes were analysed. The most prevalent aminoglycoside resistance genes were aph(3')-Ia (133 strains, 30.3%), aac(3)-IId (100 strains, 22.8%), and aac(6')-Ib-cr (52 strains, 11.8%). The most frequent associations with β-lactam resistance genes were aph(3')-Ia or aac(3)-IId with blaTEM-1 (94 and 72 strains, respectively), and aac(3)-IIa/aac(6')-Ib-cr with blaCTX-M-15/blaOXA-1 (23 strains). These results indicate a frequent association of aac(3) and aph(3') genotypes with BSBL production, and a frequent co-occurrence of aac(6') genes with ESBL production. The high rate of co-occurrence of aminoglycoside resistance and β-lactamase production must be considered in combination therapy.

A novel New Delhi metallo-β-lactamase (NDM) variant, NDM-19, was identified in a carbapenem-resistant E. coli strain isolated from a subcutaneous infection of a laparotomy scar from an Egyptian patient in a Swiss hospital. NDM-19 is a derivative of NDM-7, from which it differs by a single amino acid substitution (Ala233Val). Under zinc-limiting growth conditions, E. coli DH5α transformants producing NDM-19 displayed reduced susceptibility towards expanded-spectrum cephalosporins and carbapenems as compared to transformants producing NDM-1 or NDM-7.

Helicobacter pylori is a major human pathogen that causes a wide range of gastrointestinal pathology. Progression of H. pylori induced gastritis to more severe disease has been found to highly correlate with the array of virulence factors expressed by the pathogen. The objective of this study was twofold: first, to characterize the genetic diversity of H. pylori strains isolated from 41 non-atrophic gastritis patients in Switzerland, an issue that has not been investigated to date. And second, to assess the prevalence and sequence variation of H. pylori virulence factors (cagA, vacA, iceA and dupA) and genes encoding outer membrane proteins (OMPs, babA, babB, sabA, sabB, hopZ, hopQ and oipA) by whole genome sequencing (WGS) using an Illumina MiSeq platform. WGS identified high genetic diversity in the analyzed H. pylori strains. Most H. pylori isolates were assigned to hpEurope (95.0%, 39/41), and the remaining ones (5.0%, 2/41) to hpEastAsia, subpopulation hspEAsia. Analysis of virulence factors revealed that 43.9% of the strains were cagA-positive, and the vacA s1 allele was detected in 56.0% of the isolates. The presence of cagA was found to be significantly associated (P &lt, 0.001) with the presence of vacA s1, babA2 and hopQ allele 1 as well as expression of oipA. Moreover, we found an association between the grade of gastritis and H. pylori abundance in the gastric mucosa, respectively and the presence of cagA, vacA s1 and hopQ allele 1. Among our 41 gastritis patients, we identified seven patients infected with H. pylori strains that carried a specific combination of virulence factors (i.e cagA, vacA s1 allele and babA2 allele), recently implicated in the development of more severe gastrointestinal pathology, like peptic ulcer disease and even gastric cancer. To this end, WGS can be employed for rapid and detailed characterization of virulence determinants in H. pylori, providing valuable insights into the pathogenic capacity of the bacterium. This could ultimately lead to a higher level of personalized treatment and management of patients suffering from H. pylori associated infections.

Background Interpretative reading of antimicrobial susceptibility test (AST) results allows inferring biochemical resistance mechanisms from resistance phenotypes. For aminoglycosides, however, correlations between resistance pathways inferred on the basis of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints and expert rules versus genotypes are generally poor. This study aimed at developing and validating a decision tree based on resistance phenotypes determined by disc diffusion and based on epidemiological cut-offs (ECOFFs) to infer the corresponding resistance mechanisms in Escherichia coli. Methods Phenotypic antibiotic susceptibility of thirty wild-type and 458 aminoglycoside-resistant E. coli clinical isolates was determined by disc diffusion and the genomes were sequenced. Based on well-defined cut-offs, we developed a phenotype-based algorithm (Aminoglycoside Resistance Mechanism Inference Algorithm - ARMIA) to infer the biochemical mechanisms responsible for the corresponding aminoglycoside resistance phenotypes. The mechanisms inferred from susceptibility to kanamycin, tobramycin and gentamicin were analysed using ARMIA- or EUCAST-based AST interpretation and validated by whole genome sequencing (WGS) of the host bacteria. Findings ARMIA-based inference of resistance mechanisms and WGS data were congruent in 441/458 isolates (96·3%). In contrast, there was a poor correlation between resistance mechanisms inferred using EUCAST CBPs/expert rules and WGS data (418/488, 85·6%). Based on the assumption that resistance mechanisms can result in therapeutic failure, EUCAST produced 63 (12·9%) very major errors (vME), compared to only 2 (0·4%) vME with ARMIA. When used for detection and identification of resistance mechanisms, ARMIA resolved >95% vMEs generated by EUCAST-based AST interpretation. Interpretation This study demonstrates that ECOFF-based analysis of AST data of only four aminoglycosides provides accurate information on the resistance mechanisms in E. coli. Since aminoglycoside resistance mechanisms, despite having in certain cases a minimal effect on the minimal inhibitory concentration, may compromise the bactericidal activity of aminoglycosides, prompt detection of resistance mechanisms is crucial for therapy. Using ARMIA as an interpretative rule set for editing AST results allows for better predictions of in vivo activity of this drug class., eBioMedicine, 46, ISSN:2352-3964

Objectives Rapid detection of macrolide resistance–associated mutations in Mycoplasma pneumoniae is crucial for effective antimicrobial treatment. We evaluated the Lightmix Mycoplasma macrolide assay for the detection of point mutations at nucleotide positions 2063 and 2064 in the 23S ribosomal RNA (rRNA) gene of M. pneumoniae that confer macrolide resistance. Methods Samples from 3438 patients with a respiratory tract infection were analysed by M. pneumoniae real-time PCR, and 208 (6%) of them were tested positive. In this retrospective study, 163 M. pneumoniae real-time PCR–positive samples were analysed by the Lightmix assay, and results were compared to targeted 23S rRNA sequencing. Results Macrolide-resistant M. pneumoniae were found in 15 (9%) of 163 retrospectively analysed samples. The Lightmix assay showed a sensitivity of 100% (95% confidence interval, 78.2–100) and a specificity of 100% (95% confidence interval, 97.5–100) as the detected M. pneumoniae genotype (148 wild type and 15 non–wild type) was confirmed by 23S rRNA sequencing in all samples. Conclusions The Lightmix assay is an easy-to-use and accurate molecular test that allows rapid determination of macrolide resistance in M. pneumoniae.

Bacteria belonging to the genus; Mycobacterium; are predominantly responsible for pulmonary diseases; most notably; Mycobacterium tuberculosis; causes granulomatous pulmonary infections. Here we describe a novel slow growing mycobacterial species isolated from respiratory samples from five patients, four with underlying pulmonary disease. The isolates were characterized by biochemical and molecular techniques, including whole genome sequencing. Biochemical characteristics generally match those of; M. marinum; and; M. ulcerans; ; however, the most striking difference of the new species is its ability to grow at 37°C. The new species was found to grow in human macrophages, but not amoebae, suggesting a pathogenic rather than an environmental lifestyle. Phylogenetic analysis reveals a deep-rooting relationship to; M. marinum; and; M. ulcerans; . A complete genome sequence was obtained through combining short and long-read sequencing, providing a genome of 5.6 Mb. The genome appears to be highly intact, syntenic with that of; M. marinum; , with very few insertion sequences. A vast array of virulence factors includes 283 PE/PPE surface-associated proteins, making up 10% of the coding capacity, and 22 non-ribosomal peptide synthase clusters. A comparison of six clinical isolates from the five patients shows that they differ by up to two single nucleotide polymorphisms, suggesting a common source of infection. Our findings are in accordance with the recognition of a new taxonomic entity. We propose the name; M. basiliense; , as all isolates were found in patients from the Basel area of Switzerland.

Helicobacter pylori is a major human pathogen. Diagnosis of H. pylori infection and determination of its antibiotic susceptibility still mainly rely on culture and phenotypic drug susceptibility testing (DST) that is time-consuming and laborious. Whole genome sequencing (WGS) has recently emerged in medical microbiology as a diagnostic tool for reliable drug resistance prediction in bacterial pathogens. The aim of this study was to compare phenotypic DST results with the predictions based on the presence of genetic determinants identified in the H. pylori genome using WGS. Phenotypic resistance to clarithromycin, metronidazole, tetracycline, levofloxacin, and rifampicin was determined in 140 clinical H. pylori isolates by E-Test&reg, and the occurrence of certain single nucleotide polymorphisms (SNPs) in target genes was determined by WGS. Overall, there was a high congruence of &gt, 99% between phenotypic DST results for clarithromycin, levofloxacin, and rifampicin and SNPs identified in the 23S rRNA, gyrA, and rpoB gene. However, it was not possible to infer a resistance phenotype for metronidazole based on the occurrence of distinct SNPs in frxA and rdxA. All 140 H. pylori isolates analysed in this study were susceptible to tetracycline, which was in accordance with the absence of double or triple nucleotide substitutions in the 16S rRNA gene.

Pupylation is a bacterial ubiquitin-like protein modification pathway, which results in the attachment of the small protein Pup to specific lysine residues of cellular targets. Pup was shown to serve as a degradation signal, directing proteins toward the bacterial proteasome for turnover. Recently, it was hypothesized that pupylation and proteasomal protein degradation support the survival of Mycobacterium smegmatis (Msm) during nitrogen starvation by supplying recycled amino acids. In the present study we generated a Pup deletion strain to investigate the influence of pupylation on Msm proteome in the absence of nitrogen sources. Quantitative proteomic analyses revealed a relatively low impact of Pup on MsmΔpup proteome immediately after exposure to growth medium lacking nitrogen. Less than 5.4% of the proteins displayed altered cellular levels when compared to Msm wild type. In contrast, post 24 h of nitrogen starvation 501 proteins (41% of the total quantified proteome) of Msm pup deletion strain showed significant changes in abundance. Noteworthy, important players involved in nitrogen assimilation were significantly affected in MsmΔpup. Furthermore, we quantified pupylated proteins of nitrogen-starved Msm to gain more detailed insights in the role of pupylation in surviving and overcoming the lack of nitrogen.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway.

Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response., Scientific Reports, 7 (1), ISSN:2045-2322

Biochimica et Biophysica Acta, 1843 (1), ISSN:0006-3002, ISSN:0005-2760, ISSN:0167-4838, ISSN:0304-4157, ISSN:0005-2728, ISSN:0005-2736, ISSN:0167-4781, ISSN:0304-4165, ISSN:0925-4439, ISSN:0167-4889, ISSN:1388-1981, ISSN:1570-9639, ISSN:0304-419X, ISSN:0926-6585, ISSN:0005-2744, ISSN:0926-6593, ISSN:1874-9399, ISSN:0926-6534, ISSN:0005-2787, ISSN:0005-2795, ISSN:0304-4173, ISSN:0926-6577, ISSN:0926-6569, ISSN:0926-6542, ISSN:0926-6526, ISSN:0926

Summary Proteasome-bearing bacteria make use of a ubiquitin-like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C-terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Δdop strain, pupylation is severely impaired and the steady-state levels of two known proteasomal substrates are drastically increased. Pupylation can be re-established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C-terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N-terminal ATP-binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.

Acetogens use the Wood-Ljungdahl pathway for reduction of carbon dioxide to acetate. This pathway not only allows reoxidation of reducing equivalents during heterotrophic growth but also supports chemolithoautotrophic growth on H(2) + CO(2). The latter argues for this pathway being a source for net energy conservation, but the mechanism involved remains unknown. In addition to CO(2), acetogens can use alternative electron acceptors, such as nitrate or caffeate. Caffeate respiration in the model acetogen Acetobacterium woodii is coupled to energy conservation via a chemiosmotic mechanism, with Na(+) as coupling ion. The pathway and its bioenergetics were solved in some detail very recently. This review focuses on the regulation of caffeate respiration, describes the enyzmes involved, summarizes the evidence for a potential Na(+)-translocating ferredoxin:NAD(+)-oxidoreductase (Rnf complex) as a new coupling site, and hypothesizes on the role of this Rnf complex in the Wood-Ljungdahl pathway.

The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes H 2 -dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits α (EtfA) and β (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NAD + oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na + pump. These data suggest the following electron transport chain: H 2 → ferredoxin → NAD + → Etf → caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD + reduction catalyzed by Rnf.

A prokaryotic protein tagging system called pupylation that is analogous to ubiquitylation in eukaryotes has recently been described. In this process, prokaryotic ubiquitin-like protein (Pup) is coupled to substrate proteins via an isopeptide bond in order to target them for degradation by the proteasome. The ligation occurs via a condensation reaction involving a carboxylate of the C-terminal glutamate of Pup (located in a conserved terminal Gly-Gly-Glu motif) and the side-chain amino group of a substrate lysine. Here we have used a combination of NMR and biochemical experiments with free lysine and a physiological protein substrate (PanB) to show that the coupling occurs through the side-chain carboxylate of the glutamate in the GGE motif rather than the carboxy terminus but that the glutamate must be located at the C-terminal position to be coupled.

Post-translational modification of proteins with prokaryotic ubiquitin-like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two-step process in which the carboxy-terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ-carboxylate to substrate lysines by the Pup ligase PafA. In this study, we identify a new feature of the pupylation system by demonstrating that Dop also acts as a depupylase in the Pup proteasome system in vivo and in vitro. Dop removes Pup from substrates by specific cleavage of the isopeptide bond. Depupylation can be enhanced by the unfolding activity of the mycobacterial proteasomal ATPase Mpa.

Pupylation is the bacterial equivalent of ubiquitin conjugation, and it involves C-terminal Pup conjugation to lysines to target proteins for proteasomal degradation. This modification reaction has now been reconstituted in vitro using enzymes from the pathogen Mycobacterium tuberculosis. The Pup deamidase (Dop) of this pathway has been defined, and PafA has been shown to conjugate deamidated Pup to substrates. In analogy to ubiquitin in eukaryotes, the bacterial protein Pup is attached to lysine residues of substrate proteins, thereby targeting them for proteasomal degradation. It has been proposed that, before its attachment, Pup is modified by deamidation of its C-terminal glutamine to glutamate. Here we have identified Dop (locus tag Rv2112) as the specific deamidase of Pup in Mycobacterium tuberculosis. Deamidation requires ATP as a cofactor but not its hydrolysis. Furthermore, we provide experimental evidence that PafA (locus tag Rv2097) ligates deamidated Pup to the proteasomal substrate proteins FabD and PanB. This formation of an isopeptide bond requires hydrolysis of ATP to ADP, suggesting that deamidated Pup is activated for conjugation via phosphorylation of its C-terminal glutamate. By combining these enzymes, we have reconstituted the complete bacterial ubiquitin-like modification pathway in vitro, consisting of deamidation and ligation steps catalyzed by Pup deamidase (Dop) and Pup ligase (PafA).

ClpA is an Hsp100 chaperone that uses the chemical energy of ATP to remodel various protein substrates to prepare them for degradation. It comprises two AAA+ modules and the N-domain, which is attached N-terminally to the first AAA+ module through a linker. On the basis of cryo-electron microscopic and X-ray crystallographic data it has been suggested that the linker confers mobility to the N-domain. In order to define the role of the N-domain in ClpAP-dependent substrate degradation we have generated a ΔN variant at the protein level by introducing a protease cleavage site. The ClpA molecule generated in this way lacks the N-domain and the associated linker but is impaired only slightly in the processing of substrates that are degraded independently of ClpS. In fact, it shows increased catalytic efficiency in the degradation of ssrA-tagged GFP compared to ClpAwt. The role of the linker attaching the N-domain to the bulk of the molecule was probed by characterizing variants with different lengths of the linker. The degradation efficiency of a ClpS-dependent N-end rule substrate, FRliGFP, is reduced for linkers that are shorter or longer than natural linkers but remains the same for the variant where the linker is replaced by an engineered sequence of equivalent length. These results suggest that the flexible attachment of the N-domains to ClpA allows their recruitment to the pore on demand for certain substrates, while allowing them to move out of the way for substrates binding directly to the pore.

The anaerobic acetogenic bacterium Acetobacterium woodii can conserve energy by oxidation of various substrates coupled to either carbonate or caffeate respiration. We used a cell suspension system to study the regulation and kinetics of induction of caffeate respiration. After addition of caffeate to suspensions of fructose-grown cells, there was a lag phase of about 90 min before caffeate reduction commenced. However, in the presence of tetracycline caffeate was not reduced, indicating that de novo protein synthesis is required for the ability to respire caffeate. Induction also took place in the presence of CO 2 , and once a culture was induced, caffeate and CO 2 were used simultaneously as electron acceptors. Induction of caffeate reduction was also observed with H 2 plus CO 2 as the substrate, but the lag phase was much longer. Again, caffeate and CO 2 were used simultaneously as electron acceptors. In contrast, during oxidation of methyl groups derived from methanol or betaine, acetogenesis was the preferred energy-conserving pathway, and caffeate reduction started only after acetogenesis was completed. The differential flow of reductants was also observed with suspensions of resting cells in which caffeate reduction was induced prior to harvest of the cells. These cell suspensions utilized caffeate and CO 2 simultaneously with fructose or hydrogen as electron donors, but CO 2 was preferred over caffeate during methyl group oxidation. Caffeate-induced resting cells could reduce caffeate and also p -coumarate or ferulate with hydrogen as the electron donor. p -Coumarate or ferulate also served as an inducer for caffeate reduction. Interestingly, caffeate-induced cells reduced ferulate in the absence of an external reductant, indicating that caffeate also induces the enzymes required for oxidation of the methyl group of ferulate.

Cell suspensions of Acetobacterium woodii prepared from cultures grown on fructose plus caffeate catalyzed caffeate reduction with electrons derived from molecular hydrogen. Hydrogen-dependent caffeate reduction was strictly Na + dependent with a K m for Na + of 0.38 mM; Li + could substitute for Na + . The sodium ionophore ETH2120, but not protonophores, stimulated hydrogen-dependent caffeate reduction by 280%, indicating that caffeate reduction is coupled to the buildup of a membrane potential generated by primary Na + extrusion. Caffeate reduction was coupled to the synthesis of ATP, and again, ATP synthesis coupled to hydrogen-dependent caffeate reduction was strictly Na + dependent and abolished by ETH2120, but not by protonophores, indicating the involvement of a transmembrane Na + gradient in ATP synthesis. The ATPase inhibitor N , N ′-dicyclohexylcarbodiimide (DCCD) abolished ATP synthesis, and at the same time, hydrogen-dependent caffeate reduction was inhibited. This inhibition could be relieved by ETH2120. These experiments are fully compatible with a chemiosmotic mechanism of ATP synthesis with Na + as the coupling ion during hydrogen-dependent caffeate reduction by A . woodii .

BACKGROUND Resistance to clarithromycin in Helicobacter pylori (H pylori) is mediated by mutations in the domain V of the 23S rRNA gene (A2142G, A2143G, A2142C). Other polymorphisms in the 23S rRNA gene have been reported to cause low-level clarithromycin resistance but their importance is still under debate. In this study, we aimed to develop and evaluate the CRHP Finder webtool for detection of the most common mutations mediating clarithromycin resistance from whole-genome sequencing (WGS) data. Moreover, we included an analysis of 23 H pylori strains from Danish patients between January 2017 and September 2019 in Copenhagen, Denmark. MATERIALS AND METHODS The CRHP Finder detects the fraction of each of the four nucleotides in nucleotide positions 2142, 2143, 2182, 2244 and 2712 of the 23S rRNA gene in H pylori (E coli numbering) by aligning raw sequencing reads (fastq format) with k-mer alignment (KMA). The nucleotide distribution in each position is compared to previously described point mutations mediating clarithromycin resistance in H pylori, and a genotypic prediction of the clarithromycin resistance phenotype is presented as output. For validation of the CRHP webtool, 137 fastq paired-end sequencing datasets originating from a well-characterized strain collection of H pylori were analyzed. RESULTS The CRHP Finder correctly identified all resistance mutations reported in the sequencing data of 137 H pylori strains. In the 23 Danish H pylori strains, CRHP Finder detected A2143G (13%) in all resistant strains, and T2182C (13%) and C2244T (4,3%) nucleotide exchanges in only susceptible strains. CONCLUSION In this study, we present the validation of the first webtool for H pylori resistance prediction based on the detection of 23S rRNA mutations (A2142C, A2142G, A2143G, T2182C, C2244T, T2712C) from WGS data of H pylori.

Background Mycoplasma sp. are well recognized as etiological agents of respiratory and sexually transmitted disease. Mycoplasma penetrans, a species of Mycoplasma sp., has been frequently detected in HIV-positive patients and associated with the progression of HIV-associated disease. To date, there is only a single case report describing M. penetrans as the causative agent of a severe respiratory tract infection in a HIV-negative patient. Case presentation In this report, we describe the case of M. penetrans bacteremia in a HIV-negative, 38-year-old, female, immunocompromised, solid organ transplant patient (combined kidney and pancreas transplantation in 2016), who was admitted to our hospital with anemic uterine bleeding and fever of 38.3 °C. Several hours before her admission at our university hospital, a latex bladder catheter was inserted into her uterus and she complained about fatigue, dizziness and ongoing vaginal bleeding. Laboratory examination showed severe anemia, but microbiological examination was inconspicuous (culture negative vaginal and cervical smears, negative urine culture). Bacterial blood cultures showed a growth signal after 4 h, but microscopic examination with Gram staining and subcultures on different agar media did not identify bacterial pathogens. To identify the bacterial cause of malignancy in the patient, metagenomic sequencing of the blood culture was performed that identified M. penetrans. Conclusion Metagenomic sequencing identified M. penetrans in an immunosuppressed patient with culture-negative bacteremia. Clinicians should be aware of the opportunistic potential of M. penetrans that may cause severe infections in certain vulnerable patient populations and the limitations of culture and Gram staining for confirming the presence of fastidious bacterial pathogens like Mycoplasma spp.