Frontiers in Microbiology

Daily cotrimoxazole (TMP/SXT) prophylaxis is part of the HIV treatment package for all new HIV-infected individuals in Uganda. Although this treatment has shown reduced morbidity and mortality in HIV, it remains controversial due to its contribution to developing antibiotic-resistant bacteria. Moreover, the effects of daily use of a broad-spectrum antibiotic on the gut microbiome remain unknown. To study the early effects, we analysed shotgun metagenome sequence data from stool samples of five newly HIV-infected individuals initiating TMP/SXT prophylaxis longitudinally for the first 30 days of treatment. Using shotgun metagenomics sequencing, we generated both taxonomic and functional profiles from each patient and compared gut microbial changes Pre-TMP/SXT and post-TMP/SXT on Day 5, Day 14, and Day 30. Daily TMP/SXT prophylaxis resulted in a shift characterised by an enrichment of Prevetollea and Ruminococcus genera members and the depletion of Lactococcus and Bacteroides genera members. Furthermore, these microbial shifts were associated with changes in the functional profile revealed by a differential abundance of pathways of amino acid metabolism, carbohydrate metabolism, and nucleotide biosynthesis linked to members of the Bacteroidaceae and Enterobacteriaceae families. TMP/SXT daily prophylaxis in HIV-infected individuals is associated with dramatic changes in microbial composition and functional profiles; however, other factors such as Age, Gender, HIV clinical stage, and ART regiment are at play. Further investigation is needed to examine the implication of these shifts on clinical management and outcomes among HIV patients.

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proofs that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in A_DKE and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologs of this protein were found in other Micrarchaeota genomes, which raises the question, whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated and the Micrarchaeum expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described co-culture, containing Micrarchaeum A_DKE and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of A_DKE. Both organisms are clearly distinguishable by cell size, shape and surface structure.

Pathogenic bacteria recognize environmental cues to vary gene expression for host adaptation. Moving from ambient to host temperature, Yersinia enterocolitica responds by immediately repressing flagella synthesis and inducing the virulence plasmid (pYV)-encoded type III secretion system. In contrast, shifting from host to ambient temperature requires 2.5 generations to restore motility suggesting a link to the cell cycle. We hypothesized that differential DNA methylation contributes to temperature-regulated gene expression. We tested this hypothesis by comparing single-molecule real-time (SMRT) sequencing of Y. enterocolitica DNA from cells growing exponentially at 22{degrees}C and 37{degrees}C. The inter-pulse duration ratio rather than the traditional QV scoring was the kinetic metric to compare DNA from cells grown at each temperature. All 565 YenI restriction sites were fully methylated at both temperatures. Among the 27,118 DNA adenine methylase (Dam) sites, 42 had differential methylation patterns while 17 remained unmethylated regardless of temperature. A subset of the differentially methylated Dam sites localized to promoter regions of predicted regulatory genes including LysR-type and PadR-like transcriptional regulators, and a cyclic-di-GMP phosphodiesterase. The unmethylated Dam sites localized with a bias to the replication terminus, suggesting they were protected from Dam methylase. No cytosine methylation was detected at Dcm sites. DATA SUMMARYAll methylation/base modification data are available at figshare at https://dx.doi.org/10.6084/m9.figshare.3493247 and https://dx.doi.org/10.6084/m9.figshare.3493310. IMPACT STATEMENTOrganisms sense and respond to their environment, in part, by epigenetic variation mediated by DNA methylation. Pathogenic bacteria vary gene expression to allow survival and activate virulence systems in response to host temperature. Yersinia enterocolitica, a facultative intracellular pathogen, respond by immediately repressing flagella synthesis and inducing the virulence plasmid-encoded type III secretion system. In this work, we examined the locations of DNA methylation throughout the Y. enterocolitica genome. While most methylation target sites were fully methylated, we identified sites with disparate temperature-dependent methylation. Several of these sites were within promoter regions of predicted regulatory genes. Differences in DNA methylation in promoter sequences are often responsible for variations in transcription. Identification of these differences in methylation provide likely candidates for regulators responsible for temperature-dependent phenotypes.

Metaproteomics is as a promising technique for studying the human gut microbiota, because it can reveal the taxonomic profile and also shed light on the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing and microbial cell disruption methods for metaproteomic analyses of human gut microbiota. An unsupervised principal component analysis showed that different methods produced similar human gut microbial taxonomic profiles. An unsupervised two-way hierarchical clustering analysis identified the microbial taxonomic signatures associated with each method. Proteobacteria and Bacteroidetes identification was favored by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads. Ascomycota identification was enhanced by using large-sized glass beads during sample processing for stool dispersion. Euryarchaeota identification was improved with a combination of small and medium-sized glass beads for cell disruption. Assessments of the relative abundance of Firmicutes, Actinobacteria and Spirochaetes improved when ultrasonication was performed before cell disruption with glass beads. The latter method also increased the overall number of identified proteins. Taxonomic and protein functional analyses of metaproteomic data derived from stool samples from six healthy individuals showed common taxonomic profiles. We also detected certain proteins involved in microbial functions relevant to the host and related mostly to particular taxa, such as B12 biosynthesis and short chain fatty acid production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. Finally, in this metaproteomic study we identified several human proteins, mostly related to the anti-microbial response, which could contribute to determining the beneficial and detrimental relationships between gut microbiota and human cells in particular human diseases.

Scanning electron microscopy (SEM) is widely used to investigate bacterial surface architecture; however, sample preparation protocols may introduce structural artefacts that compromise the interpretation of morphometric and ultrastructural features. This issue becomes especially relevant for extremophilic microorganisms, whose specialized cell envelopes may respond differently to chemical fixation. In this study, we evaluated the effects of two aldehyde-based fixation protocols, 2.5% glutaraldehyde and Karnovskys solution, combined with different fixation times (1, 3, and 24 h) and the presence or absence of osmium tetroxide (OsO4) post-fixation, on three Gram-positive bacterial strains: the polyextremophiles Exiguobacterium sp. S17 and Nesterenkonia sp. Act20, and the mesophile Kocuria rosea CH-021. Morphological preservation was assessed using morphometric parameters, including cellular area and surface-to-volume ratio, together with texture analysis based on Haralick descriptors derived from grey-level co-occurrence matrices (GLCM). Results showed that fixation conditions significantly affected morphometric and textural features in a strain-dependent manner. Although overall morphology appeared preserved, quantitative analyses revealed marked differences in surface texture and structural integrity among treatments. Osmium tetroxide modified morphometric and textural parameters, although its effects varied among strains and fixation conditions. These findings highlight the importance of integrated quantitative approaches combining cellular geometry and surface texture analysis for the evaluation of SEM preparation protocols in structurally specialized microorganisms.

Bacterial vaginosis (BV) is a common infection of the lower genital tract with a vaginal microbiome dysbiosis caused by decreasing of lactobacilli. Previous studies suggested that supplementation with live Lactobacillus may benefit the recovery of BV, while the outcomes vary in people from different regions. Herein, we aim to evaluate the effectiveness of oral Chinese-origin Lactobacillus with adjuvant metronidazole (MET) on treating Chinese BV patients. In total, 67 Chinese women with BV were enrolled in this parallel controlled trial and randomly assigned to two study groups: a control group treated with MET vaginal suppositories for 7 days and a probiotic group treated with oral Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 as an adjuvant to MET for 30 days. By comparing the participants with Nugent scores [&ge;] 7 and < 7 on days 14, 30, and 90, we found that oral administration of probiotics did not improve BV cure rates (57.14% and 67.74% at day 14, 57.14% and 58.06% at day 30, 32.14% and 48.39% at day 90 for probiotic and control group respectively). However, the probiotics were effective in restoring vaginal health after cure by showing higher proportion of participants with Nugent scores < 4 in the probiotic group compared to the control group (87.50% and 71.43% on day 14, 93.75% and 88.89% on day 30, and 77.78% and 66.67% on day 90). The relative abundance of the probiotic strains was significantly increased in the gut microbiome of the probiotic group compared to the control group at day 14, but no significance was detected after 30 and 90 days. Also, the probiotics were not detected in vaginal microbiome, suggesting that L. gasseri TM13 and L. crispatus LG55 mainly acted through the gut. A higher abundance of Prevotella timonensis at baseline was significantly associated with long-term cure failure of BV and greatly contributed to the enrichment of the lipid IVA synthesis pathway, which could aggravate inflammation response. To sum up, L. gasseri TM13 and L. crispatus LG55 can restore the vaginal health of patients recovering from BV, and individualized intervention mode should be developed to improve BV cure rates.

The expression of dctA, encoding the aerobic C4-dicarboxylate (C4-DC) transporter DctA of Escherichia coli, and its use in the presence of alternative carbon sources was characterized. dctA is regulated by cAMP-CRP and substrates that control cAMP levels, either through the phosphotransferase system (PTS), or through their metabolic link to PEP synthesis. The data indicates that phosphorylation of the regulator EIIAGlc of the glucose-specific PTS represents the mediator for regulation. The dctA promotor region contains a class I CRP-binding site (position -81.5) and a DcuR-binding site (position -105.5). The response regulator DcuR of the C4-DC-activated DcuS-DcuR two-component system is known to stimulate expression of dctA, and cAMP-CRP is known to stimulate expression of dcuS-dcuR. Thus, activation of dctA expression by cAMP-CRP and DcuR is organized in a coherent feed-forward loop (FFL) where cAMP-CRP positively regulates the expression of dctA by direct stimulation and by stimulating the expression of dcuR. Stimulation by DcuR is presumed to require DNA bending by cAMP-CRP. In this way, CRP-FFL integrates carbon catabolite control and C4-DC-specific regulation. Moreover, EIIAGlc of the glucose-specific PTS strongly interacts with DctA, which could lead to substrate exclusion of C4-DCs when preferred carbon substrates such as sugars are present. Since C4-DCs are perceived in the periplasmic space by the sensor DcuS, the substrate exclusion is not linked to inducer exclusion, contrasting classical inducer exclusion known for the lactose permease LacY. Thus, aerobic C4-DC metabolism is tightly regulated at the transcriptional and post-translational levels, whereas uptake of L-aspartate by DcuA is essentially unaffected. Overall, transcriptional and post-translational regulation of dctA expression and DctA function efficiently fine-tunes C4-DC catabolism in response to other preferred carbon sources.

The Lobaria pulmonaria holobiont comprises algal, fungal, cyanobacterial, and bacterial components. We investigated L. pulmonarias bacterial microbiome in the adaptation of this ecologically sensitive lichen species to diverse climatic conditions. Our central hypothesis posited that microbiome composition and functionality aligns with continental-scale climatic parameters related to temperature and precipitation. We also tested the impact of short-term weather dynamics, sampling season, and algal/fungal genotypes on microbiome variation. Metaproteomics provided insights into compositional and functional changes within the microbiome. Climatic variables explained 41.64% of microbiome variation, surpassing the combined influence of local weather and sampling season at 31.63%. Notably, annual mean temperature and temperature seasonality emerged as significant climatic drivers. Microbiome composition correlated with algal, not fungal genotype, suggesting similar environmental recruitment for the algal partner and microbiome. Differential abundance analyses revealed distinct protein compositions in sub-atlantic lowland and alpine regions, indicating differential microbiome responses to contrasting environmental/climatic conditions. Proteins involved in oxidative and cellular stress were notably different. Our findings highlight microbiome plasticity in adapting to stable climates, with limited responsiveness to short-term fluctuations, offering new insights into climate adaptation in lichen symbiosis.

The determinants and consequences of drought-related shrub mortality were studied for over a decade, as a model for desertification processes, in a semi-arid long-term ecological research station. Recent studies have shown that geodiversity is an important spatial predictor of plant viability under extreme drought conditions. Homogeneous hillslopes, with a deep soil profile and lack of stoniness, could not support shrubs under long term drought conditions due to low water storage in their soil. Conversely, heterogeneous hillslopes, with shallow soil profiles and high stoniness, supported shrub communities under similar conditions, due to the comparatively greater soil-water content. In the current study, we investigated the effect of hillslope geodiversity on the soil microbial diversity. Using DNA metabarcoding, we found small but consistent differences in the microbial community compositions of the homogeneous and heterogeneous hillslopes; more ammonia oxidizing and reducing-sugar degrading bacteria are found in the homogeneous hillslopes, possibly dwindling the ammonia supply to shrubs. Additionally, based on functional metagenomic reconstruction, we suggest that homogeneous hillslopes have lower superoxide and antibiotics production, leading to reduced protection against pathogens. In fungi, we observed an increase in possible pathogens, at the expense of lichen forming fungi. Lichens are considered to support soil-water by slowly releasing intercepted raindrops. In conclusion, we show that not only plant-diversity but also microbial-diversity is shaped by geodiversity, and that the community shift in homogeneous hillslopes may further promote shrub mortality in this drought-prone, water limited ecosystem. HIGHLIGHTSO_LIHomogeneous hillslopes reduce soil water storage and increase aeration. C_LIO_LIAmmonia oxidizers and reducing-sugar degraders dwindle ammonia supply for plants. C_LIO_LIHomogenous hillslopes do not support moisture providing lichens. C_LIO_LIReduced antibiotics and superoxide secretion capacitate pathogens. C_LIO_LIGeodiversity facilitates microbial regulation during drought. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/434393v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1503208org.highwire.dtl.DTLVardef@4c589org.highwire.dtl.DTLVardef@144d17dorg.highwire.dtl.DTLVardef@1159040_HPS_FORMAT_FIGEXP M_FIG C_FIG

The halophilic archaeon Haloferax mediterranei is a promising candidate for polyhydroxyalkanoate production, offering several advantages due to its extremophilic physiology. While its primary polyhydroxyalkanoate synthase, a class III enzyme composed of PhaCHme and PhaEHme subunits, has been well characterized, the genome encodes three additional phaC paralogs (phaC1, phaC2 and phaC3), which were previously labeled as cryptic and remain poorly understood. In this study, we systematically investigated these paralogs by employing a targeted bioinformatics pipeline, revealing notable diversity in polyhydroxyalkanoate synthases among Halobacteriales and underscoring the distinctiveness of H. mediterranei. We further analyzed the native transcriptional expression profiles of all phaC paralogs under three physiologically relevant conditions: growth-limiting and growth-permissive conditions, as well as valeric acid supplementation to alter polyhydroxyalkanoate monomer composition. RT-qPCR analysis demonstrated that all three paralogs are transcriptionally active and differentially expressed, refuting earlier assumptions of their cryptic nature. Expression patterns were found not to correlate to polymer composition but to be dependent on growth phase, suggesting a potential physiological role for each paralog in native polyhydroxyalkanoate metabolism. These findings offer new insights into the functional complexity of polyhydroxyalkanoate biosynthesis in H. mediterranei and lay the groundwork for future metabolic engineering aimed at optimizing biopolymer production.

Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase and error-free template switching catalyzed by RecA, and their competition on the arrested fork affects bacterial SOS mutagenesis. DnaE2 is an error-prone TLS polymerase, and its functions require ImuA and ImuB. Here we investigated the function of imuA, imuB and dnaE2 in Myxococcus xanthus and found that imuA showed differences from imuB and dnaE2 in bacterial growth, resistance and mutation frequency. Transcriptomics analysis found that ImuA were associated with bacterial SOS response. Yeast-two-hybrid scanning revealed that ImuA interacted with RecA1 besides ImuB. Protein activity analysis proved that ImuA had no DNA binding activity, but inhibited the DNA binding and recombinase activity of RecA1. These findings highlight that ImuA not only participates in TLS by binding ImuB, but also inhibits the recombinase activity of RecA1 in M. xanthus, suggesting a role of ImuA in the two replication restart pathways. ImportanceDnaE2 is responsible for bacterial SOS mutagenesis in nearly one third of sequenced bacterial strains. However, its mechanism, especially the function of its accessory protein ImuA, is still unclear. Here we reported that M. xanthus ImuA might facilitate DnaE2 TLS by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the scientific problem of choosing one of the two restart pathways to repair the stalled replication fork.

Mycobacterium tuberculosis (Mtb) poses a significant global health burden. Rv0647c, an essential Mtb cell wall protein, is a potential drug target. We studied its Mycolicibacterium smegmatis (MSMEG) homologue, MSMEG_1353, using a CRISPRi conditional knockdown. MSMEG_1353 depletion increased cell width and volume, delayed log-phase initiation, slowed aggregation, reduced biofilm formation, and heightened susceptibility to antibiotics and sodium dodecyl sulfate (SDS). AlphaFold, sequence alignment, and UNIPROT analyses suggest MSMEG_1353 functions as a protein kinase, with conserved residues in intermediate high-confidence regions potentially forming an ATP-binding site. Investigating its role in cell envelope biosynthesis, mass spectrometry revealed elevated levels of mycolic acid biosynthesis proteins upon MSMEG_1353 knockdown. RT-qPCR confirmed upregulation of the fabD-acpM-kasA-KasB-accD6 operon, encoding key mycolic acid synthesis enzymes. Notably, a strong negative correlation with MSMEG_0911, the predominant isocitrate lyase, important in the glyoxylate cycle, was observed via both MS and RT-qPCR. Collectively, MSMEG_1353 deficiency compromises cell wall integrity, likely due to altered lipid composition resulting from dysregulated mycolic acid biosynthesis and lipid metabolism. These findings support the development of models explaining MSMEG_1353s involvement in these pathways.

Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness to process-specific parameters. Here, in order to build synthetic genetic circuits that increase bacterial resistance to diverse stress conditions, we mined novel stress tolerance genes from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized ten novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.

Bacteria have evolved numerous regulatory pathways to survive in changing environments. The SOS response is an inducible DNA damage repair system that plays an indispensable role in bacterial adaptation and pathogenesis. Here we report a discovery of the previously uncharacterized protein Lmo0946 as an SOS response interfering factor (Sif) in the human pathogen Listeria monocytogenes. Functional genetic studies demonstrated that sif is indespensible for normal growth of L. monocytogenes in stress-free as well as multi-stress conditions, and sif contributes to susceptibility to {beta}-lactam antibiotics, biofilm formation and virulence. Absence of Sif promoted the SOS response and elevated expression of mobilome genes accompanied by mobilization of the A118 prophage and ICELm-1 mobile genetic elements (MGEs). These changes were found to be associated with decreased expression of general stress response genes from the {sigma}B regulon as well as virulence genes, including the PrfA regulon. Together, this study uncovers an unexpected role of a previously uncharacterized factor, Sif, as an inhibitor of the SOS response in L. monocytogenes. SUMMARYThis study uncovers an unexpected role of a previously uncharacterized factor, Sif, as an inhibitor of the SOS response in L. monocytogenes.

Upon infection, Mycobacterium tuberculosis (M.tb) reaches the alveolar space and comes in close contact with human alveolar lining fluid (ALF) for an uncertain period of time prior to its encounter with alveolar cells. We showed that homeostatic ALF hydrolytic enzymes modify the M.tb cell envelope, driving M.tb-host cell interactions. Still, the contribution of ALF during M.tb infection is poorly understood. Here, we exposed 4 M.tb strains with different levels of virulence, transmissibility, and drug resistance (DR) to physiological concentrations of human ALF for 15-min and 12-h, and performed RNA sequencing. Gene expression analysis showed a temporal and strain-specific adaptation to human ALF. Differential expression (DE) of ALF-exposed vs. unexposed M.tb revealed a total of 397 DE genes associated with lipid metabolism, cell envelope and processes, intermediary metabolism and respiration, and regulatory proteins, among others. Most DE genes were detected at 12-h post-ALF exposure, with DR-M.tb strain W-7642 having the highest number of DE genes. Interestingly, genes from the KstR2 regulon, which controls the degradation of cholesterol C and D rings, were significantly upregulated in all strains post-ALF exposure. These results indicate that M.tb-ALF contact drives initial metabolic and physiologic changes in M.tb, with potential implications in infection outcome. IMPORTANCETuberculosis, caused by airborne pathogen Mycobacterium tuberculosis (M.tb), is one of the leading causes of mortality worldwide. Upon infection, M.tb reaches the alveoli and gets in contact with human alveolar lining fluid (ALF), where ALF hydrolases modify the M.tb cell envelope driving subsequent M.tb-host cell interactions. Still, the contributions of ALF during infection are poorly understood. We exposed 4 M.tb strains to ALF for 15-min and 12-h and performed RNA sequencing, demonstrating a temporal and strain-specific adaptation of M.tb to ALF. Interestingly, genes associated with cholesterol degradation were highly upregulated in all strains. This study shows for the first time that ALF drives global metabolic changes in M.tb during the initial stages of the infection, with potential implications in disease outcome. Biologically relevant networks and common and strain-specific bacterial determinants derived from this study could be further investigated as potential therapeutic candidates.

Bacterial second messenger signaling often plays an important role in cellular physiology. In this study, we have attempted to understand how c-di-AMP synthesis and degradation are transcriptionally regulated in M. smegmatis. We have discovered that, although c-di-AMP synthesis gene disA exists in a multi-gene operon; a sub-operon promoter arrangement plays a key role under various stress conditions, keeping its dual function property intact. Further, we learned that c-di-AMP plays a role in the autoregulation of the disA promoter to limit intracellular c-di-AMP concentration. We also identified an alternate start codon within the disA gene which can lead to the synthesis of truncated DisA protein at times using an independent stress-inducible promoter. All in all, this study was helpful to understand how c-di-AMP synthesis is regulated under normal and stress conditions linked to its physiological relevance in M. smegmatis.

The physiological mechanisms behind the emergence of antibiotic-resistant bacteria from their antibiotic-persister population are beginning to be explored. Here we delineate the sequential physiological events that drive the emergence of rifampin-resistant rpoB mutants from rifampin-persister population of mycobacteria during prolonged exposure. The rifampin-persister population generated elevated levels of hydroxyl radical, which inflicted mutations, enabling regrowth of the persister cells to form multi-septated, multi-nucleated elongated cells. These cells, through multiple divisions, produced multiple sister-daughter cells, causing an abrupt, unexpectedly high increase of rifampin-resistant colonies. Similar response was observed against moxifloxacin also. Earlier studies on the rifampin/moxifloxacin-exposed laboratory/clinical M. tuberculosis strains from in-vitro cultures and infected mouse-lung also showed cfu spurt, but went unnoticed/unreported. It is likely that these sequential physiological events may be driving the emergence of antibiotic-resistant tubercle bacilli in TB patients also. Escherichia coli also has been found to respond similarly against subminimal inhibitory concentrations of ciprofloxacin. Thus, the present findings attain broad significance as a general physiological mechanism used by diverse bacterial genera to emerge as drug-resistant strains against antibiotics.

Newly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.

Coxiella burnetii (Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a "Reverse evolution" approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media.

In present study, we describe "tat-fimbriae (tafi)" - a novel type of archaeal surface appendages isolated from haloarchaeon Haloarcula hispanica. These filamental structures are unique because they are formed of protein subunits secreted through the twin-arginine translocation pathway (Tat-pathway), in contrast to well-known archaeal surface filamentous structures secreted by the general secretory pathway (Sec-pathway). No cases of the role of Tat-pathway in the assembly of archaeal and bacterial filamentous structures have been described to date. "Tafi" are the first example of such structures. The precursor of the major tafi protein subunit TafA contains the N-terminal signal peptide carrying a twin-arginine consensus motif and fimbria-forming mature TafA lacks this signal peptide. We analyzed the gene neighborhood of the tafA homologues in the known haloarchaeal genomes and found a conservative cluster of seven associated genes tafA, B, C, D, E, F, G. We assume that all of them take part in the tafi synthesis. TafC and TafE proteins, whose precursor sequences also contain twin-arginine motifs, were detected as minor components of tafi. TafE protein is structurally similar to TafA, while TafC contains a TafA-like N-terminal domain and a C-terminal "laminin G-like" domain capable of functioning as an adhesin. TafD is annotated as a signal peptidase I. The functions of TafB, TafF and TafG are not known yet. This study demonstrated that {Delta}tafA and {Delta}tafD deletion mutant strains synthesized archaella and not tafi, and only tafi were detected in {Delta}arlK (gene of common archaellin/pilin signal peptidase) deletion strain. It was shown that the expression of complete Har. hispanica taf-gene cluster in a heterologous host Haloferax volcanii that does not have similar genes leads to synthesis of recombinant tafi structures similar to the native ones. The tafi function remains elusive, but our preliminary data suggest that these structures may be involved in cell adhesion to different surfaces or substrates.