Following a proposal to emend Appendix 9 of the International Code of Nomenclature of Prokaryotes with guidelines for the naming of genera after geographical locations, I here report the outcome of the ballot on this proposal by the members of the International Committee on Systematics of Prokaryotes and present the guidelines to be incorporated in Appendix 9.

In this paper the Judicial Commission provides general guidance for interpreting the International Code of Nomenclature of Prokaryotes (ICNP) and specific assistance to authors, reviewers and editors of a Request for an Opinion, or of other suggestions related to the ICNP. The role of the Judicial Commission is recapitulated, particularly with respect to the processing of such Requests. Selected kinds of nomenclature-related proposals are discussed that are unsuitable as the basis for a Request. Particular emphasis is put on Requests for placing names or epithets on the list of nomina rejicienda, and a dichotomous identification key is provided to guide potential authors of a Request that targets the name of a species or subspecies because of issues with its type strain. To this end, the criteria for the valid publication of such names under the ICNP are revisited. Aspects of other kinds of Requests are also addressed. The study is based on a comprehensive review of all Judicial Opinions issued since the publication of the Approved Lists in 1980. One goal of this paper is to assist potential authors in deciding whether their concern should be the subject of a Request, and if so, in composing it with the greatest chance of success. It is also clarified how to obtain additional help regarding nomenclature-related issues.

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor $\\sigma$ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with $\\sigma$ and the RNAP $\\beta$ subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

The genus \'Candidatus Phytoplasma\' was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus \'Ca. Phytoplasma\', the proposed guidelines were revised and clarified to accommodate those \'Ca. Phytoplasma\' species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing <98.65 % sequence identity with the reference strain but >98.65 % with other strain(s) within the same \'Ca. Phytoplasma\' species should be considered related strains to that \'Ca. Phytoplasma\' species. The guidelines herein, keep the original published reference strains. However, to improve \'Ca. Phytoplasma\' species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of \'Ca. Phytoplasma\' species and alternative reference strains described are reported. For new \'Ca. Phytoplasma\' species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published \'Candidatus Phytoplasma\' species, including \'Ca. P. cocostanzaniae\' and \'Ca. P. palmae\' described in this manuscript.

In this study, it was aimed to investigate Mycobacterium bovis strains isolated from lungs and lymph nodes of slaughtered animals on clonal level by using different methods such as spoligotyping, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNAs (RAPD-PCR) and OUT-PCR. Comparative evaluation of these methods was further conducted.
A total of 38 M. bovis isolates were evaluated in the study. DNA isolation of all M. bovis strains isolated from pruvat free Löwenstein Jensen medium was done by boiling method for ERIC-PCR, RAPD-PCR, and OUT PCR. Mickle device was used for DNA isolation for spoligotyping method.
In 38 M. bovis isolates examined in our study, 4 different groups were determined by spoligotyping and RAPD-PCR test methods, and 5 different groups were detected in ERIC-PCR tests. In the OUT-PCR tests, the band which provides sufficient type separation was not observed.
ERIC-PCR, RAPD-PCR, and OUT-PCR methods are easily applicable, simple, and relatively inexpensive methods for evaluating the differences between origins in the typing of M. bovis. The tests need to be evaluated in more detail with extensive studies.

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPo). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyse RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, \'bind-unwind-load-and-lock\', model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

No previous studies have validated current clinical practice guidelines for the management of non-blanching rashes in children who have received meningococcal B and C vaccinations. The aim of this study was to evaluate the performance of existing clinical practice guidelines in the diagnosis of invasive meningococcal disease in children presenting with a fever and non-blanching rash in the UK.
The Petechiae in Children (PiC) study was a prospective, multicentre cohort study involving children (aged <18 years) presenting to 37 paediatric emergency departments in the UK with a fever (≥38°C) and a new-onset non-blanching rash or features suggestive of meningococcal infection. Children with pre-existing haematological conditions (ie, haematological malignancy, idiopathic thrombocytopenic purpura, or coagulopathy) or an existing diagnosis of Henoch-Schonlein purpura were excluded. Invasive meningococcal disease was confirmed by positive culture or a quantitative PCR test for Neisseria meningitidis from either blood or cerebrospinal fluid samples. The primary outcome was the performance of six tailored clinical practice guidelines from participating centres (London, Nottingham, Newcastle-Birmingham-Liverpool, Glasgow, Chester, and Bristol) and two clinical practice guidelines from the National Institutes for Health and Care Excellence (NICE; CG102 and NG51) in identifying children with invasive meningococcal disease, assessed by the sensitivity and specificity of each clinical practice guideline. This study is registered with ClinicalTrials.gov, NCT03378258.
Between Nov 9, 2017, and June 30, 2019, 1513 patients were screened, of whom 1329 were eligible and were included in the analysis. The median age of patients was 24 months (IQR 12-48). 1137 (86%) of 1329 patients had a blood test and 596 (45%) received parenteral antibiotics. 19 (1%) patients had confirmed meningococcal disease. All eight clinical practice guidelines had a sensitivity of 1·00 (95% CI 0·82-1·00) for identifying meningococcal disease. The specificities of NICE guidelines CG102 (0·01 [95% CI 0·01-0·02]) and NG51 (0·00 [0·00-0·00]) for identifying meningococcal disease were significantly lower than that of tailored clinical practice guidelines (p<0·0001). The best performing clinical practice guidelines for identifying meningococcal disease were the London (specificity 0·36 [0·34-0·39]) and Nottingham (0·34 [0·32-0·37]) clinical practice guidelines.
Invasive meningococcal disease is a rare cause of non-blanching rashes in children presenting to the emergency department in the UK. Current NICE guidelines perform poorly when compared with tailored clinical practice guidelines. These findings suggest that UK national guidance could be improved by shifting towards a tailored approach.
Public Health Agency.

Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.

Consensus-based protein engineering strategy has been applied to various proteins and it can lead to the design of proteins with enhanced biological performance. Histone-like HUs comprise a protein family with sequence variety within a highly conserved 3D-fold. HU function includes compacting and regulating bacterial DNA in a wide range of biological conditions in bacteria. To explore the possible impact of consensus-based design in the thermodynamic stability of HU proteins, the approach was applied using a dataset of sequences derived from a group of 40 mesostable, thermostable, and hyperthermostable HUs. The consensus-derived HU protein was named HUBest, since it is expected to perform best. The synthetic HU gene was overexpressed in E. coli and the recombinant protein was purified. Subsequently, HUBest was characterized concerning its correct folding and thermodynamic stability, as well as its ability to interact with plasmid DNA. A substantial increase in HUBest stability at high temperatures is observed. HUBest has significantly improved biological performance at ambience temperature, presenting very low Kd values for binding plasmid DNA as indicated from the Gibbs energy profile of HUBest. This Kd may be associated to conformational changes leading to decreased thermodynamic stability and, therefore, higher flexibility at ambient temperature.

CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a \"directionality\" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.