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Summary: This special issue of Molecular Microbiology marks the 25th anniversary of the discovery of the extracytoplasmic function (ECF) σ factors, proteins that subsequently emerged as the largest group of alternative σ factors and one of the three major pillars of signal transduction in bacteria, alongside one‐ and two‐component systems. A single bacterial genome can encode > 100 ECF σ factors, and combined with their cognate anti‐σ factors, they represent a modular design that primarily functions in transmembrane signal transduction. Here, we first describe the immediate events that led to the 1994 publication in the Proceeding of the National Academy of Sciences USA, and then set them in the broader context of key events in the history of σ biology research. [ABSTRACT FROM AUTHOR]

In silico analyses identified a Crp/Fnr family transcription factor (HcpR) in sulfate-reducing bacteria that controls expression of the hcp gene, which encodes the hybrid cluster protein and contributes to nitrosative stress responses. There is only one hcpR gene in the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, but two copies in Desulfovibrio desulfuricans 27774, which can use nitrate as an alternative electron acceptor to sulfate. Structures of the D. desulfuricans hcpR1, hcpR2 and hcp operons are reported. We present evidence that hcp expression is regulated by HcpR2, not by HcpR1, and that these two regulators differ in both their DNA-binding site specificity and their sensory domains. HcpR1 is predicted to be a b-type cytochrome. HcpR1 binds upstream of the hcpR1 operon and its synthesis is regulated coordinately with hcp in response to NO. In contrast, hcpR2 expression was not induced by nitrate, nitrite or NO. HcpR2 is an iron-sulfur protein that reacts with NO and O2. We propose that HcpR1 and HcpR2 use different sensory mechanisms to regulate subsets of genes required for defense against NO-induced nitrosative stress, and that diversification of signal perception and DNA recognition by these two proteins is a product of D. desulfuricans adaptation to its particular environmental niche. [ABSTRACT FROM AUTHOR]

Vibrio mimicus is a gram-negative bacterium responsible for diseases in humans. Three strains of V. mimicus identified as V. mimicus 87, V. mimicus 92 and V. mimicus 93 were isolated from a shrimp processing facility in Guaymas, Sonora, Mexico. The strains were analyzed using several molecular techniques and according to the cluster analysis they were different, their similarities ranged between 51.3% and 71.6%. ERIC-PCR and RAPD (vmh390R) were the most discriminatory molecular techniques for the differentiation of these strains. The complete genomes of two strains (V. mimicus 87, renamed as CAIM 1882, and V. mimicus 92, renamed as CAIM 1883) were sequenced. The sizes of the genomes were 3.9 Mb in both strains, with 2.8 Mb in ChI and 1.1 Mb in ChII. A 12.7% difference was found in the proteome content (BLAST matrix). Several virulence genes were detected (e.g. capsular polysaccharide, an accessory colonization factor and genes involved in quorum-sensing) which were classified in 16 categories. Variations in the gene content between these genomes were observed, mainly in proteins and virulence genes (e.g., hemagglutinin, mobile elements and membrane proteins). According to these results, both strains were different, even when they came from the same source, giving an insight of the diversity of V. mimicus. The identification of various virulence genes, including a not previously reported V. mimicus gene (acfD) in ChI in all sequenced strains, supports the pathogenic potential of this species. Further analysis will help to fully understand their potential virulence, environmental impact and evolution. [ABSTRACT FROM AUTHOR]

The emergence of carbapenemase-producing Gram-negative bacilli is a worldwide problem. To date, no study has evaluated the prevalence of faecal carriage of carbapenemase-producing and carbapenem-resistant Gram-negative bacilli (CR GNB) in France. From 1 February to 30 April 2012, we conducted a prospective, multicentre study in three University Hospitals and four General Hospitals in the south of France. The carriage of carbapenemase-producing Enterobacteriaceae (CPE) and other CR GNB was screened by both cultivation on chromID® CARBA and chromID® OXA-48 media (bioMérieux) and molecular tools [multiplex polymerase chain reaction (PCR) and NucliSENS EasyQ® KPC (bioMérieux)]. The genetic relationship between isolates was assessed by rep-PCR (DiversiLab, bioMérieux) or multilocus sequence typing (MLST). The prevalences of CR GNB and carbapenemase-producing bacteria were 2.4 % (27/1,135) and 0.4 % ( n = 5), respectively. Two strains corresponded to OXA-23-producing Acinetobacter baumannii and belonged to the widespread sequence type (ST) 2/international clone II, whereas one strain was an ST15 OXA-48-producing Klebsiella pneumoniae. Two OXA-48-producers were detected exclusively by PCR. This first French study revealed the very low dissemination of carbapenemase-producing bacteria in patients attending hospitals in southern France during a non-outbreak situation. However, the increasing description of epidemic cases in this area must reinforce the use of hygiene procedures to prevent diffusion of these multidrug-resistant microorganisms. [ABSTRACT FROM AUTHOR]

Metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa is a growing issue across the globe. Fast and reliable diagnostic tools are needed for appropriate implementation of infection control measures. In this study we evaluated the performance of three commercial combined disk tests, two EDTA based in-house combined disk tests and the Carba NP test in comparison to molecular detection of MBL genes on 133 meropenem non-susceptible non-duplicate P. aeruginosa clinical isolates. The meropenem/DPA based commercial KPC + MBL-confirm ID kit (Rosco Diagnostica, Denmark) and the MASTDISCS™ ID carbapenemase (Enterobacteriaceae) detection disc set (MAST Diagnostics, UK) showed sensitivities of 31.1 % and 28.8 % and specificities of 69.3 % and 79.6 %, respectively. The total MBL confirm kit (Rosco Diagnostica, Denmark) contains imipenem/DPA and imipenem/EDTA combination disks. Evaluation of the single disk combinations revealed 84.4 % sensitivity and 81.8 % specificity for the imipenem/DPA assay and 86.7 % sensitivity and 51.1 % specificity for the imipenem/EDTA test. Applying both tests simultaneously resulted in a slightly higher sensitivity of 88.9 % but a lower specificity of 48.9 % when compared to the single tests alone. The Carba NP test showed 93.3 % sensitivity and 96.6 % specificity. All phenotypic combined disk tests lacked either sensitivity or specificity for the detection of MBL in P. aeruginosa. The Carba NP test showed excellent test properties, but suffers from drawbacks in handling and high costs. The optimal diagnostic approach needs to be chosen depending on the epidemiological situation, laboratory resources and availability of molecular confirmation tests. [ABSTRACT FROM AUTHOR]

We explore the genetic diversity of class D oxacillinases, including OXA-23, -24 (-40), -58 and, particularly, the intrinsic OXA-51-like genes, among multidrug-resistant (MDR) Acinetobacter baumannii strains from inpatients at a tertiary care hospital in Riyadh, Saudi Arabia. Sequence-based typing (SBT) of the OXA-51-like gene was carried out on 253 isolates. Selected isolates ( n = 66) were subjected to multilocus sequence typing (MLST). The polymerase chain reaction (PCR) typing results showed that all isolates ( n = 253) contained the OXA-51-like and OXA-23 genes. However, the OXA-58 gene was detected in five isolates. Further, none of the isolates had the OXA-40 (identical to the OXA-24) gene. SBT revealed a high OXA-51-like genotypic diversity and showed that all isolates were clustered into four main groups: OXA-66 (62.3 %), followed by OXA-69 (19.1 %), OXA-132 (7.6 %) and other OXA-51-like genes (10.3 %), including OXA-79, -82, -92, -131 and -197. MLST revealed four main sequence types (STs), 2, 19, 20 and 25, among the isolates, in addition to six isolates with newly designated ST194-ST197 singletons. Further, a high prevalence (81.4 %) of OXA-66 and OXA-69-like genes in A. baumannii was identified. More studies are essential in order to explore the molecular mechanisms that confer carbapenem-resistant phenotypes for A. baumannii isolates and to investigate the genetic diversity of other OXA-D genes. [ABSTRACT FROM AUTHOR]

Pseudogenes are defined as non-functional relatives of genes whose protein-coding abilities are lost and are no longer expressed within cells. They are an outcome of accumulation of mutations within a gene whose end product is not essential for survival. Proper investigation of the procedure of pseudogenization is relevant for estimating occurrence of duplications in genomes. Frankineae houses an interesting group of microorganisms, carving a niche in the microbial world. This study was undertaken with the objective of determining the abundance of pseudogenes, understanding strength of purifying selection, investigating evidence of pseudogene expression, and analysing their molecular nature, their origin, evolution and deterioration patterns amongst domain families. Investigation revealed the occurrence of 956 core pFAM families sharing common characteristics indicating co-evolution. WD40, Rve_3, DDE_Tnp_IS240 and phage integrase core domains are larger families, having more pseudogenes, signifying a probability of harmful foreign genes being disabled within transposable elements. High selective pressure depicted that gene families rapidly duplicating and evolving undoubtedly facilitated creation of a number of pseudogenes in Frankineae. Codon usage analysis between protein-coding genes and pseudogenes indicated a wide degree of variation with respect to different factors. Moreover, the majority of pseudogenes were under the effect of purifying selection. Frankineae pseudogenes were under stronger selective constraints, indicating that they were functional for a very long time and became pseudogenes abruptly. The origin and deterioration of pseudogenes has been attributed to selection and mutational pressure acting upon sequences for adapting to stressed soil environments. [ABSTRACT FROM AUTHOR]

Many hemipterans are associated with symbiotic bacteria, which are usually found intracellularly in specific bacteriomes. In this study, we provide the first molecular identification of the bacteriome-associated, obligate endosymbiont in a Gondwanan relict insect taxon, the moss bugs ( Hemiptera: Coleorrhyncha: Peloridiidae), which represents one of the oldest lineages within the Hemiptera. Endosymbiotic associations of fifteen species of the family were analysed, covering representatives from South America, Australia/ Tasmania and New Zealand. Phylogenetic analysis based on four kilobases of 16 S-23 S r RNA gene fragments showed that the obligate endosymbiont of Peloridiidae constitute a so far unknown group of Gammaproteobacteria which is named here ' Candidatus Evansia muelleri'. They are related to the sternorrhynchous endosymbionts Candidatus Portiera and Candidatus Carsonella. Comparison of the primary-endosymbiont and host ( COI + 28 S rRNA) trees showed overall congruence indicating co-speciation the hosts and their symbionts. The distribution of the endosymbiont within the insect body and its transmission was studied using FISH. The endosymbionts were detected endocellularly in a pair of bacteriomes as well as in the 'symbiont ball' of the posterior pole of each developing oocyte. Furthermore, ultrastructural analysis of the Malpighian tubules revealed that most host nuclei are infected by an endosymbiotic, intranuclear bacterium that was determined as an Alphaproteobacterium of the genus Rickettsia. [ABSTRACT FROM AUTHOR]

Campylobacter is genetically highly diverse and undergoes frequent intraspecific recombination. Turkeys have been identified as an important reservoir for Campylobacter jejuni which is of public health significance. The assessment of the genetic diversity among Campylobacter population is critical for our understanding of the epidemiology of this bacterium. The genetic profiles were different according to the molecular typing methods used. The performance of established flaA genotyping, multilocus sequencing typing (MLST) and DNA microarray assay based on the ArrayTube™ technology was evaluated using 14 Campylobacter jejuni isolated from a commercial turkey flock. The flaA typing was performed using PCR-RFLP with restriction enzymes Sau3AI, AluI, a ‘composite’ flaA analysis of AluI and Sau3AI and DdeI. The 14 isolates were differentiated into 3, 5, 7 and 9 genotypes, respectively. Entire flaA gene and short variable region (SVR) sequences were analysed. Sequencing of the entire flaA provided 11 different genotypes. flaA-SVR sequence analysis detected 8 flaA alleles and 4 flaA peptides. One new flaA allele type (528) was identified. MLST analysis represented 10 different sequence types (STs) and 5 clonal complexes (CCs). The microarray assay recognised 14 different genotypes. The discriminatory indices were 0.560, 0.802, 0.857, and 0.912 for flaA-RFLP depending on the used enzymes, 0.890 for flaA-SVR, 0.967 for entire flaA sequencing, 0.945 for MLST and 1.00 for the DNA microarray assay. The flaA gene was genetically stable over 20 passages on blood agar. In conclusion, the different typing tools demonstrated a high level of genetic heterogeneity of Campylobacter jejuni in a turkey flock, indicating that a single flock can be infected by multiple genotypes within one rearing cycle. DNA microarray-based assays had the highest discriminatory power when compared with other genotyping tools. [ABSTRACT FROM AUTHOR]

Abstract: The use of 16S rRNA gene sequencing within the regulatory workflow may help to reduce the time and labor involved in the identification and differentiation of Salmonella enterica isolates. However, a comprehensive, standardized reference library is needed in order to use this method with regulatory samples. The goal of this project was to acquire 16S rRNA partial and full gene sequences for a variety of S. enterica isolates and to use these sequences to build a custom 16S rRNA reference library. A total of 535 S. enterica isolates representing over 100 serotypes and 5 subspecies were selected for 16S rRNA partial gene sequencing (~500bp) and 66 isolates representing 32 serotypes and 2 subspecies were selected for 16S rRNA full gene sequencing (~1500bp). PCR, sequencing, and automated sequence assembly and editing were carried out using the MicroSEQ ID Microbial Identification System (Applied Biosystems). High quality sequences were obtained for 94.4% and 95.5% of the isolates sequenced over the partial and full genes, respectively. These sequences did not show sufficient divergence to reliably differentiate serotypes; however, they could be differentiated using 16S rRNA sequence typing based on intragenomic heterogeneity. A total of 83 unique 16S sequence types were obtained for use in the partial gene library and 58 unique 16S sequence types were obtained for entry into the full gene library. Preliminary sequencing results with one isolate analyzed in replicate were promising, with consistent matches to a specific 16S type in the custom library. The result of this study is a custom S. enterica 16S rRNA type library for potential use in the identification of isolates at the species, subspecies, and molecular subtype level. Further work will include validating the method for parameters such as exclusivity, sensitivity, and reproducibility. [Copyright &y& Elsevier]

Considering the major involvement of gut microflora in the digestive function of various macro-organisms, bacterial communities inhabiting fish guts may be the main actors of organic matter degradation by fish. Nevertheless, the extent and the sources of variability in the degradation potential of gut bacterial communities are largely overlooked. Using Biolog Ecoplate™ and denaturing gradient gel electrophoresis ( DGGE), we explored functional (i.e. the ability to degrade organic matter) and genetic (i.e. identification of DGGE banding patterns) diversity of fish gut bacterial communities, respectively. Gut bacterial communities were extracted from fish species characterized by different diets sampled along a salinity gradient in the Patos- Mirim lagoons complex ( Brazil). We found that functional diversity was surprisingly unrelated to genetic diversity of gut bacterial communities. Functional diversity was not affected by the sampling site but by fish species and diet, whereas genetic diversity was significantly influenced by all three factors. Overall, the functional diversity was consistently high across fish individuals and species, suggesting a wide functional niche breadth and a high potential of organic matter degradation. We conclude that fish gut bacterial communities may strongly contribute to nutrient cycling regardless of their genetic diversity and environment. [ABSTRACT FROM AUTHOR]

Abstract: Marine unicellular cyanobacteria, represented by Synechococcus and Prochlorococcus, dominate the total phytoplankton biomass and production in oligotrophic ocean. In this study, we employed comparative genomics approaches to extensively investigate synonymous codon usage bias and evolutionary rates in a large number of closely related species of marine unicellular cyanobacteria. Although these two groups of marine cyanobacteria have a close phylogenetic relationship, we find that they are highly divergent not only in codon usage patterns but also in the driving forces behind the diversification. It is revealed that in Prochlorococcus, mutation and genome compositional constraints are the main forces contributing to codon usage bias, whereas in Synechococcus, translational selection. In addition, nucleotide substitution rate analysis indicates that they are not evolving at a constant rate after the divergence and that the average d N/d S values of core genes in Synechococcus are significantly higher than those in Prochlorococcus. Our evolutionary genomic analysis provides the first insight into codon usage, evolutionary genetic mechanisms and environmental adaptation of Synechococcus and Prochlorococcus after divergence. [Copyright &y& Elsevier]

Enteric bacterial pathogens are known to effectively pass through the extremely acidic mammalian stomachs and cause infections in the small and/or large intestine of human hosts. However, their acid-survival strategy and pathogenesis mechanisms remain elusive, largely due to the lack of tools to directly monitor and manipulate essential components (e.g., defense proteins or invasive toxins) participating in these processes. Herein, we have extended the pyrrolysine-based genetic code expansion strategy for encoding unnatural amino acids in enteric bacterial species, including enteropathogenic Escherichia coli, Shigella, and Salmonella. Using this system, a photo-cross-linking amino acid was incorporated into a Shigella acid chaperone HdeA (shHdeA), which allowed the identification of a comprehensive list of in vivo client proteins that are protected by shHdeA upon acid stress. To further demonstrate the application of our strategy, an azide-bearing amino acid was introduced into a Shigella type 3 secretion effector, OspF, without interruption of its secretion efficiency. This site-specifically installed azide handle allowed the facile detection of OspF's secretion in bacterial extracellular space. Taken together, these bioorthogonal functionalities we incorporated into enteric pathogens were shown to facilitate the investigation of unique and important proteins involved in the pathogenesis and stress-defense mechanisms of pathogenic bacteria that remain exceedingly difficult to study using conventional methodologies. [ABSTRACT FROM AUTHOR]

The twin-arginine translocation (Tat) pathway is found in bacteria, archaea, and plant chloroplasts, where it is dedicated to the transmembrane transport of fully folded proteins. These proteins contain N-terminal signal peptides with a specific Tat-system binding motif that is recognized by the transport machinery. In contrast to other protein transport systems, the Tat system consists of multiple copies of only two or three usually small (∼8-30 kDa) membrane proteins that oligomerize to two large complexes that transiently interact during translocation. Only one of these complexes includes a polytopic membrane protein, TatC. The other complex consists of TatA. Tat systems of plants, proteobacteria, and several other phyla contain a third component, TatB. TatB is evolutionarily and structurally related to TatA and usually forms tight complexes with TatC. Minimal two-component Tat systems lacking TatB are found in many bacterial and archaeal phyla. They consist of a 'bifunctional' TatA that also covers TatB functionalities, and a TatC. Recent insights into the structure and interactions of the Tat proteins have various important implications. [ABSTRACT FROM AUTHOR]

Abstract: Enterotoxigenic Escherichia coli (ETEC) produces a variety of colonization factors necessary for attachment to the host cell, among which CS6 is one of the most prevalent in ETEC isolates from developing countries. The CS6 operon is composed of 4 genes, cssA, cssB, cssC, and cssD. The molecular mechanism of CS6 assembly and cell surface presentation, and the contribution of each protein to the attachment of the bacterium to intestinal cells remain unclear. In the present study, a series of css gene-deletion mutants of the CS6 operon were constructed in the ETEC genetic background, and their effect on adhesion to host cells and CS6 assembly was studied. Each subunit deletion resulted in a reduction in the adhesion to intestinal cells to the same level of laboratory E. coli strains, and this effect was restored by complementary plasmids, suggesting that the 4 proteins are necessary for CS6 expression. Bacterial cell fractionation and western blotting of the mutant strains suggested that the formation of a CssA–CssB–CssC complex is necessary for recognition by CssD and transport of CssA–CssB to the outer membrane as a colonization factor. [Copyright &y& Elsevier]

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Previously, we demonstrated the reductive dehalogenation of dichlorobenzene (DCB) isomers to monochlorobenzene (MCB), and MCB to benzene in sediment microcosms derived from a chlorobenzene-contaminated site. In this study, enrichment cultures were established for each DCB isomer and each produced MCB and trace amounts of benzene as end products. MCB dehalogenation activity could only be transferred in sediment microcosms. The 1,2-DCB-dehalogenating culture was studied the most intensively. Whereas Dehalococcoides spp. were not detected in any of the microcosms or cultures, Dehalobacter spp. were detected in 16S rRNA gene clone libraries from 1,2-DCB enrichment cultures, and by PCR using Dehalobacter-specific primers in 1,3-DCB and 1,4-DCB enrichments and MCB-dehalogenating microcosms. Quantitative PCR showed Dehalobacter 16S rRNA gene copies increased up to 3 orders of magnitude upon dehalogenation of DCBs or MCB, and that nearly all of bacterial 16S rRNA genes in a 1,2-DCB-dehalogenating culture belonged to Dehalobacter spp. Dehalobacter 16S rRNA genes from DCB enrichment cultures and MCB-dehalogenating microcosms showed considerable diversity, implying that 16S rRNA sequences do not predict dehalogenation-spectra of Dehalobacter spp. These studies support a role for Dehalobacter spp. in the reductive dehalogenation of DCBs and MCB, and this genus should be considered for its potential impact on chlorobenzene fate at contaminated sites. [ABSTRACT FROM AUTHOR]

Objectives We characterized plasmids encoding CTX-M-14 β-lactamase originating from Escherichia coli isolates recovered from patients with uncomplicated cystitis or individuals with faecal colonization in Hong Kong from 2002 to 2004. Methods Plasmids carrying CTX-M-14 were studied by conjugation, replicon typing, S1 nuclease-PFGE and plasmid PCR-restriction fragment length polymorphism (RFLP). The complete sequence of pHK01, a 70 kb plasmid encoding CTX-M-14 from an E. coli strain, was determined and the results compared with reference plasmids and aligned with GenBank data. Results The blaCTX-M-14 plasmids could be transferred in 23 of 44 E. coli strains tested. Among the 23 transconjugants, the replicon types of the CTX-M-14-encoding plasmid were FII (n = 13), I1-Iγ (n = 4), F1B (n = 2), FII and I1-Iγ (n = 1), K (80 kb, n = 1) and undetermined (n = 2). Plasmid pHK01 (FII replicon) shares a high degree of homology with R100 except mainly for a 11 kb variable region containing blaCTX-M-14 (with an upstream ISEcp1 and a downstream truncated IS903), an iron transport system, an outer membrane protein (malB, maltoporin) and a putative toxin-antitoxin plasmid stability system (yacABC). It was highly related to blaCTX-M-14 (pKF3-70) and blaCTX-M-24 (pEG356) plasmids reported from mainland China in 2006 and Vietnam in 2007, respectively. Subtyping by a plasmid PCR-RFLP scheme showed that 10 of the 13 FII plasmids originating from isolates collected by multiple laboratories exhibited either identical or highly similar profiles. Conclusions This study showed that narrow host-range FII plasmids play important roles in the dissemination of CTX-M-14. FII plasmids closely related to pHK01 have disseminated widely in the Hong Kong community. [ABSTRACT FROM PUBLISHER]

CRISPR (cluster of regularly interspaced palindromic repeats)/Cas and CRISPR/Cmr systems of Sulfolobus, targeting DNA and RNA respectively of invading viruses or plasmids are complex and diverse. We address their classification and functional diversity, and the wide sequence diversity of RAMP (repeat-associated mysterious protein)-motif containing proteins encoded in Cmr modules. Factors influencing maintenance of partially impaired CRISPR-based systems are discussed. The capacity for whole CRISPR transcripts to be generated despite the uptake of transcription signals within spacer sequences is considered. Targeting of protospacer regions of invading elements by Cas protein–crRNA (CRISPR RNA) complexes exhibit relatively low sequence stringency, but the integrity of protospacer-associated motifs appears to be important. Different mechanisms for circumventing or inactivating the immune systems are presented. [ABSTRACT FROM AUTHOR]

Initiation is a critical step in translation, during which the ribosome lands on the start codon and sets the correct reading frame for mRNA decoding. The rate and efficiency of translation are largely determined by initiation, which is therefore the preferred target of translation regulation mechanisms. Initiation has incurred an extensive evolutionary divergence among the primary domains of cell descent. The Archaea, albeit prokaryotes, have an initiation mechanism and apparatus more complex than those of the Bacteria; the molecular details of archaeal initiation are just beginning to be unravelled. The most notable aspects of archaeal initiation are the presence of two, perhaps three, distinct mechanisms for mRNA–ribosome interaction and the presence of a relatively large set of IFs (initiation factors), several of which are shared exclusively with the Eukarya. Among these, the protein termed a/eIF2 (archaeal/eukaryotic IF2) and aIF6 (archaeal IF6) are of special interest, since they appear to play key regulatory roles in the Eukarya. Studies of the function of these factors in Archaea have uncovered new features that will help to elucidate their conserved and domain-specific functions. [ABSTRACT FROM AUTHOR]

The process of information exchange between two homologous DNA duplexes is known as homologous recombination (HR) or double-strand break repair (DSBR), depending on the context. HR is the fundamental process underlying the genome shuffling that expands genetic diversity (for example during meiosis in eukaryotes). DSBR is an essential repair pathway in all three domains of life, and plays a major role in the rescue of stalled or collapsed replication forks, a phenomenon known as recombination-dependent replication (RDR). The process of HR in the archaea is gradually being elucidated, initially from structural and biochemical studies, but increasingly using new genetic systems. The present review focuses on our current understanding of the structures, functions and interactions of archaeal HR proteins, with an emphasis on recent advances. There are still many unknown aspects of archaeal HR, most notably the mechanism of branch migration of Holliday junctions, which is also an open question in eukarya. [ABSTRACT FROM AUTHOR]

Motility is a common behaviour in prokaryotes. Both bacteria and archaea use flagella for swimming motility, but it has been well documented that structures of the flagellum from these two domains of life are completely different, although they contribute to a similar function. Interestingly, information available to date has revealed that structurally archaeal flagella are more similar to bacterial type IV pili rather than to bacterial flagella. With the increasing genome sequence information and advancement in genetic tools for archaea, identification of the components involved in the assembly of the archaeal flagellum is possible. A subset of these components shows similarities to components from type IV pilus-assembly systems. Whereas the molecular players involved in assembly of the archaeal flagellum are being identified, the mechanics and dynamics of the assembly of the archaeal flagellum have yet to be established. Recent computational analysis in our laboratory has identified conserved highly charged loop regions within one of the core proteins of the flagellum, the membrane integral protein FlaJ, and predicted that these are involved in the interaction with the assembly ATPase FlaI. Interestingly, considerable variation was found among the loops of FlaJ from the two major subkingdoms of archaea, the Euryarchaeota and the Crenarchaeota. Understanding the assembly pathway and creating an interaction map of the molecular players in the archaeal flagellum will shed light on the details of the assembly and also the evolutionary relationship to the bacterial type IV pili-assembly systems. [ABSTRACT FROM AUTHOR]

Bacterial populations frequently act as a collective by secreting a wide range of compounds necessary for cell-cell communication, host colonization and virulence. How such behaviours avoid exploitation by spontaneous 'cheater' mutants that use but do not contribute to secretions remains unclear. We investigate this question using Pseudomonas aeruginosa swarming, a collective surface motility requiring massive secretions of rhamnolipid biosurfactants. We first show that swarming is immune to the evolution of rhlA'cheaters'. We then demonstrate that P. aeruginosa resists cheating through metabolic prudence: wild-type cells secrete biosurfactants only when the cost of their production and impact on individual fitness is low, therefore preventing non-secreting strains from gaining an evolutionary advantage. Metabolic prudence works because the carbon-rich biosurfactants are only produced when growth is limited by another growth limiting nutrient, the nitrogen source. By genetically manipulating a strain to produce the biosurfactants constitutively we show that swarming becomes cheatable: a non-producing strain rapidly outcompetes and replaces this obligate cooperator. We argue that metabolic prudence, which may first evolve as a direct response to cheating or simply to optimize growth, can explain the maintenance of massive secretions in many bacteria. More generally, prudent regulation is a mechanism to stabilize cooperation. [ABSTRACT FROM AUTHOR]

Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding. [ABSTRACT FROM AUTHOR]

The Cas/CRISPR system of prokaryotes is a recently recognized defence system that protects cells from invasion by foreign nucleic acids, such as plasmids or bacteriophages. Using the archaeon Sulfolobus, Gudbergsdottir et al. have demonstrated that when foreign DNA is both a target of the CRISPR system and also carries a gene that is essential for cell growth, surviving cells display mutations in the CRISPR system that eliminate the target directed spacer. This simulates natural scenarios where the invading DNA cannot be readily destroyed, such as a temperate bacteriophage that manages to evade CRISPR attack and integrate into the genome, so becoming 'self'. CRISPR deletions ranged from precise removal of a single spacer up to elimination of an entire CRISPR array but the mechanism involved is not understood. Another surprising feature was that from one to two base mutations in the same target protospacer did not affect recognition by the Sulfolobus CRISPR system, something unexpected from studies in bacteria where spacer and target protospacer must match exactly. This study shows that even though the CRISPR system is a powerful weapon it can also be a dangerous one for the cell that carries it. [ABSTRACT FROM AUTHOR]

The flagellar genes in Salmonella enterica are expressed in a temporal hierarchy that mirrors the assembly process itself. The σ-FlgM regulatory circuit plays a key role in controlling this temporal hierarchy. This circuit ensures that the class 3 genes are expressed only when the hook-basal body (HBB), a key intermediate in flagellar assembly, is complete. In this work, we investigated the role of the σ-FlgM regulatory circuit in controlling the timing and magnitude of class 3 gene expression using a combination of mathematical modelling and experimental analysis. Analysis of the model predicted that this circuit continuously controls class 3 gene expression in response to HBB abundance. We experimentally validated these predictions by eliminating different components of the σ-FlgM regulatory system and also by rewiring the transcriptional hierarchy. Based on these results, we conclude that the σ-FlgM regulatory circuit continuously senses the HBB assembly process and regulates class 3 gene expression and possibly flagellar numbers in response. [ABSTRACT FROM AUTHOR]

The importance of methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase in bacteria has started to be appreciated only in the past decade. A comprehensive analysis of its various roles here demonstrates that it is an integral component of the activated methyl cycle, which recycles adenine and methionine through S-adenosylmethionine (SAM)-mediated methylation reactions, and also produces the universal quorum-sensing signal, autoinducer-2 (AI-2). SAM is also essential for synthesis of polyamines, N-acylhomoserine lactone (autoinducer-1), and production of vitamins and other biomolecules formed by SAM radical reactions. MTA, SAH and 5′-deoxyadenosine (5′dADO) are product inhibitors of these reactions, and are substrates of MTA/SAH nucleosidase, underscoring its importance in a wide array of metabolic reactions. Inhibition of this enzyme by certain substrate analogues also limits synthesis of autoinducers and hence causes reduction in biofilm formation and may attenuate virulence. Interestingly, the inhibitors of MTA/SAH nucleosidase are very effective against the Lyme disease causing spirochaete, Borrelia burgdorferi, which uniquely expresses three homologous functional enzymes. These results indicate that inhibition of this enzyme can affect growth of different bacteria by affecting different mechanisms. Therefore, new inhibitors are currently being explored for development of potential novel broad-spectrum antimicrobials. [ABSTRACT FROM AUTHOR]

The FNR protein in Escherichia coli is an O sensor that modifies global gene expression to adapt the cell to anaerobic growth. Regulation of FNR involves continuous cycling of the protein between its active and inactive states under aerobic conditions without apparent function. This raises the question of what benefit to the overall life cycle might compensate for the cost of cycling and reveals that the role of this process is poorly understood. To address this problem, we introduce the concept of a 'system design space', which provides a rigorous definition of phenotype at the molecular level and a means of visualizing the phenotypic repertoire of the system. Our analysis reveals undesirable and desirable phenotypes with an optimal constellation of parameter values for the system. To facilitate a more concrete understanding of what the design space represents, we analyse mutations that alter the apparent dimerization rate constant of FNR. We show that our estimated wild-type value of this rate constant, which is difficult to measure in situ, is located within this constellation and that the behaviour of the system is compromised in mutants if the value of the apparent dimerization rate constant lies beyond the bounds of this optimal constellation. [ABSTRACT FROM AUTHOR]

Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi. [ABSTRACT FROM AUTHOR]

In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis ( Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild-type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for egulated n opper epressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host. [ABSTRACT FROM AUTHOR]

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism. [ABSTRACT FROM AUTHOR]

Ornate, large, extremophilic (OLE) RNAs are large, non-coding transcripts characterized by their ornate secondary structure and presence predominantly in Gram-positive, extremophilic bacteria. A gene for an OLE-associated protein (OAP) is almost always located immediately downstream of the OLE gene. OAP has no extensive homology to other proteins and is predicted to form multiple transmembrane domains. We show that this protein forms a ribonucleoprotein complex with OLE RNA using at least 2:1 protein : RNA stoichiometry. A series of truncated OLE RNA constructs was used to establish that most of the RNA can be deleted without eliminating protein binding. Two primary binding sites are present within the RNA, although additional binding determinants exist and extensive structural stabilization is induced by OAP. RNA fluorescence in situ hybridization (FISH) was used in Escherichia coli to demonstrate that ribonucleoprotein complex formation localizes the RNA near cell membranes of this heterologous system. Therefore, the majority of the complex structure formed by OLE RNA may perform a biochemical function that requires membrane localization. [ABSTRACT FROM AUTHOR]

The pioABC operon is required for phototrophic iron oxidative (photoferrotrophic) growth by the α-proteobacterium Rhodopseudomonas palustris TIE-1. Expression analysis of this operon showed that it was transcribed and translated during anaerobic growth, upregulation being observed only under photoferrotrophic conditions. Very low levels of transcription were observed during aerobic growth, suggesting expression was induced by anoxia. The presence of two canonical FixK boxes upstream of the identified pioABC transcription start site implicated FixK as a likely regulator. To test this possibility, a ΔfixK mutant of R. palustris TIE-1 was assessed for pioABC expression. pioABC expression decreased dramatically in ΔfixK versus WT during photoferrotrophic growth, implying that FixK positively regulates its expression; coincidently, the onset of iron oxidation was prolonged in this mutant. In contrast, pioABC expression increased in ΔfixK under all non-photoferrotrophic conditions tested, suggesting the presence of additional levels of regulation. Purified FixK directly bound only the proximal FixK box in gel mobility-shift assays. Mutant expression analysis revealed that FixK regulates anaerobic phototrophic expression of other target genes with FixK binding sites in their promoters. This study shows that FixK regulates key iron metabolism genes in an α-proteobacterium, pointing to a departure from the canonical Fur/Irr mode of regulation. [ABSTRACT FROM AUTHOR]

This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the Academy of Sciences of the Czech Republic (AS CR) [formerly Czechoslovak Academy of Sciences (CAS)] over more than 50 years. Contribution of the founder of the Institute, academician Ivan Málek, to the introduction of these topics into the scientific program of the Institute of Microbiology and to further development of these studies is also included. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: To study the frequency and diversity of class 1 integrons lacking the 3′-conserved segment (CS) in intI1-positive Escherichia coli isolates of different origins. Methods: The presence of intI1 was previously detected in 84 E. coli isolates of food (21 isolates), animal (32) and healthy-human volunteer (31) origins. The qacEΔ1-sul1 genes were analyzed by PCR and those isolates that lacked these genes were included in this work. The genetic structure of class 1 integrons was determined, using the PCR and sequencing primer-walking strategy. Isolates and plasmids were typed. Results: Class 1 integrons lacking the 3′-CS were found in 13 of the 84 intI1-positive E. coli isolates (15.5%) of food, animal, and human origins. All 13 isolates showed unrelated patterns by REP-PCR. The following gene cassette arrangements were identified inside the class 1 integrons of these 13 strains: dfrA1; dfrA5; dfrA12-orfF-aadA2-cmlA1-aadA1-qacH-IS440-sul3; dfrA12-orfF-aadA2-cmlA1-aadA1-IS440-sul3; estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3; and a new arrangement estX-psp-aadA2-cmlA1Δ-IS1294-ΔcmlA1-aadA1-qacH-IS440-sul3 that contain the IS1294 into the cmlA1 gene (included in GenBank, number EU704128). Complete or truncated mef(B) gene was detected upstream of sul3 gene in this type of integrons. Plasmids were identified in four of the studied strains by PCR-replicon-typing, detecting different combinations of IncY, I1, FIC, FII, FIB plasmids. Non-classic integrons were located into plasmids of 100–150kb in four studied strains. Conclusions: Occurrence and diversity of class 1 integrons lacking 3′-CS among the studied intI1-positive E. coli isolates of different origins were relatively high. The sul3 gene was detected in most of class 1 integrons lacking 3′-CS. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterial interspersed repetitive units and variable number tandem repeats typing (MIRU–VNTR) is a useful technique that has been recently applied to characterize members of the Mycobacterium avium complex (MAC). The aim of this study was to examine the genetic variability among a collection of Spanish M. avium subspecies paratuberculosis (M. a. paratuberculosis) isolates with a combination of MIRU–VNTR loci. For this purpose we tested six MIRU–VNTR loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32, VNTR-292 and VNTR-259) in 70 M. a. paratuberculosis isolates of Types II and III that were recovered from 22 Spanish localities during a nine-year period (1998–2007). The combination of five loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32 and VNTR-259) enabled the differentiation of 12 allelic profiles, with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.84. Moreover, we obtained MIRU–VNTR patterns that were unique for each of the M. a. paratuberculosis types analyzed (II and III); other patterns were host-related or restricted to geographic areas. Therefore, this MIRU–VNTR approach could be a useful sub-typing molecular tool in order to get a better sense of the epidemiology of Johne''s disease. [Copyright &y& Elsevier]

In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into α-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5α proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process. [ABSTRACT FROM AUTHOR]

For some time, post-translational small protein modifications were found only in eukaryotes; much later, such modifications were identified in some species of bacteria. The recent discovery of ubiquitin-like proteins that form polymeric chains and covalently modify proteins in archaea finally closes the evolutionary gap among the domains of life. [Copyright &y& Elsevier]

Abstract: To clarify the molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli, the murA, glpT, uhpT, uhpA, ptsI and cyaA genes were sequenced from six fosfomycin-resistant isolates. Two strains were found to harbour a mutation in the murA gene that leads to an amino acid substitution (Asp369Asn or Leu370Ile) in the target protein. The remaining four strains carried specific mutations in the glpT gene; one strain possessed a mutation and the other three strains possessed truncated versions of the GlpT transporter owing either to the presence of insertion sequences or a deletion in the coding region of the gene. Two of the strains with truncated GlpT had also lost the entire uhpT gene, which encodes another fosfomycin transporter. Uptake of specific substrates for the transporters was either totally blocked or reduced in strains possessing truncated forms of GlpT or those lacking the uhpT gene. Escherichia coli strains expressing an amino-acid-substituted MurA were at least eight-fold more resistant to fosfomycin than the strain overproducing wild-type MurA. In conclusion, novel amino acid substitutions in MurA or the loss of function of transporters were identified as mechanisms of fosfomycin resistance in clinical isolates of E. coli. [Copyright &y& Elsevier]

This review concerns how Escherichia coli detects environmental inorganic orthophosphate (Pi) to regulate genes of the phosphate (Pho) regulon by the PhoR/PhoB two-component system (TCS). Pi control by the PhoR/PhoB TCS is a paradigm of a bacterial signal transduction pathway in which occupancy of a cell surface receptor(s) controls gene expression in the cytoplasm. The Pi signaling pathway requires seven proteins, all of which probably interact in a membrane-associated signaling complex. Our latest studies show that Pi signaling involves three distinct processes, which appear to correspond to different states of the sensory histidine kinase PhoR: an inhibition state, an activation state, and a deactivation state. We describe a revised model for Pi signal transduction of the E. coli Pho regulon. [Copyright &y& Elsevier]

Two-component signal transduction systems are abundant in prokaryotes. They enable cells to adjust multiple cellular functions in response to changing environmental conditions. These systems are also found, although in much smaller numbers, in lower eukaryotes and plants, where they appear to control a few very specific functions. Two-component systems have evolved in Bacteria from much simpler one-component systems bringing about the benefit of extracellular versus intracellular sensing. We review reports establishing the origins of two-component systems and documenting their occurrence in major lineages of Life. [Copyright &y& Elsevier]

Summary: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) expressing Panton-Valentine leukocidin (PVL) have been characterized worldwide, but it has not been done in Egypt. In this study, we analyzed the molecular characteristics of PVL+CA-MRSA in Egypt, compared their genetic patterns with that of PVL+ methicillin-susceptible S. aureus and PVL+CA-MRSA from different countries, and investigated the allelic variations among their lukS-PV and lukF-PV gene sequences. The prevalence of PVL+MRSA was 19.04%. They belonged to different genetic clones with multi-locus sequence types (MLST) 30, 80, and the novel type 1010. ST80 strains showed unique antibiotic resistance profile that was distinguishable from the European ST80 clone: they had no resistance to tetracycline and fusidic acid. Two nonsynonymous substitutions in the lukS-PV gene region and two haplotype variants among these isolates were detected. We revealed that PVL is a good marker for CA-MRSA infections and that PVL+ isolates belonged to different genotypes. [Copyright &y& Elsevier]

Mycobacterium tuberculosis is the causative agent of the global tuberculosis epidemic. To combat this successful human pathogen we need a better understanding of the basic biology of mycobacterial pathogenesis. The use of mycobacterial model systems has the potential to greatly facilitate our understanding of how M. tuberculosis causes disease. Recently, studies using mycobacterial models, including M. bovis BCG, M. marinum, and M. smegmatis have significantly contributed to understanding M. tuberculosis. Specifically, there have been advances in genetic manipulation of M. tuberculosis using inducible promoters and recombineering that alleviate technical limitations in working with mycobacteria. Model systems have helped elucidate how secretion systems function at both the molecular level and during virulence. Mycobacterial models have also led to interesting hypotheses about how M. tuberculosis mediates latent infection and host response. While there is utility in using model systems to understand tuberculosis, each of these models represent distinct mycobacterial species with unique environmental adaptations. Directly comparing findings in model mycobacteria to those in M. tuberculosis will illuminate the similarities and differences between these species and increase our understanding of why M. tuberculosis is such a potent human pathogen. [Copyright &y& Elsevier]

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 Δ dop 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. [ABSTRACT FROM AUTHOR]

σE, one of the best characterized mycobacterial extracytoplasmic function sigma factors, is involved in virulence, surface stress response and modulation of the inflammatory response during infection. The regulation of its activity is very complex and involves transcriptional, translational and post-translational control. Post-translational regulation is controlled by RseA, an anti-σ factor belonging to the zinc-associated anti-σ factor family. In this issue of Molecular Microbiology, Barik et al. demonstrate that RseA is a redox-sensing protein that is able to bind σE only in reducing environment. Importantly, they describe a novel positive feedback loop responsible for σE release and activation following surface stress, due to ClpC1P2-dependent proteolytic degradation of RseA, depending on its phosphorylation by the eukaryotic-like Ser/Thr protein kinase PknB. [ABSTRACT FROM AUTHOR]

The bacterial RNA polymeras holoenzyme consists of a catalytic core enzyme in complex with a σ factor that is required for promoter-specific transcription initiation. During initiation, members of the σ70 family of σ factors contact two conserved promoter elements, the −10 and −35 elements, which are separated by ∼17 base pairs (bp). σ70 family members contain four flexibly linked domains. Two of these domains, σ2 and σ4, contain determinants for interactions with the promoter −10 and −35 elements respectively. σ2 and σ4 also contain core-binding determinants. When bound to core the inter-domain distance between σ2 and σ4 matches the distance between promoter elements separated by ∼17 bp. Prior work indicates that during early elongation the nascent RNA-assisted displacement of σ4 from core can enable the holoenzyme to adopt a configuration in which σ2 and σ4 are bound to ‘promoter-like’ DNA elements separated by a single base pair. Here we demonstrate that holoenzyme can also adopt configurations in which σ2 and σ4 are bound to ‘promoter-like’ DNA elements separated by 0, 2 or 3 bp. Thus, our findings suggest that displacement of σ4 from core enables the RNA polymerase holoenzyme to adopt a broad range of ‘elongation-specific’ configurations. [ABSTRACT FROM AUTHOR]

Abstract: CcrM is one of the solitary bacterial DNA methyltransferases which does not have corresponding restriction enzymes. We established a stable ccrM-overexpressing mutant of Mesorhizobium loti, MlccrM-OX, and performed molecular and phenotypic characterization of this strain. In the M. loti MlccrM-OX infected plants, nodulation was apparently delayed at 7days after inoculation (dai), however, the nodules that eventually formed on the MlccrM-OX roots showed nitrogen fixing ability by at least 21dai. These results suggest that the initial morphogenic events were affected by ccrM-overexpression and that the correct pattern of DNA methylation of the bacterial genome is not essential for plant–microbe symbiosis, but are required for efficient nodulation. [Copyright &y& Elsevier]

During the last few years, genome-related information has become available for many microorganisms, including important food-related bacteria. Lactic acid bacteria (LAB) are important industrially in the production of fermented foods such as dairy products, sausages, sourdoughs, and vegetables. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as flavor and texture. In the present study, a species-independent functional gene microarray was developed that targets 406 genes that play key roles in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in the stress response. Also, genes linked to negative traits, such as antibiotic resistance and virulence, are represented. As LAB ecosystems contain a variety of species, there was a more global focus on these specific functional properties. Thus, an algorithm was used to design gene-specific oligonucleotides that preferably hybridize with multiple LAB species, thereby allowing controlled cross-hybridization. For proof of concept, the microarray composed of 2,269 30-mer oligonucleotides focused on LAB species that are prevalent in sourdough ecosystems. Validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides, showed that there were wide ranges in intensity and high reproducibility between microarrays. [ABSTRACT FROM AUTHOR]