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bioRxiv2017

Experimental evolution and genome data analysis of Candida albicans reveals cryptic bacteria in single yeast colonies

At least 25% of patients with positive Candida albicans bloodstream infection also have one or more bacterial species associated with the infection. These polymicrobial infections are usually caused by coagulase-negative staphylococci, most commonly Staphylococcus epidermidis and are associated with significantly worse clinical outcomes as compared to monomicrobial infections. Here we show bacteria are present in C. albicans cultures started from isolated single colony platting. These co-evolving bacteria can only be detected by the use of specific selective medium and/or long periods of incubation from 8 days up to 48 weeks (approximately 4,000 generations), used in experimental evolution methods. The detection of these co-evolving bacteria is highly dependent on the type of enzyme used for 16S rRNA gene amplification and is often missed in clinical laboratory analysis because of short incubation periods, media and temperatures, used in mycology clinical routine, that are unfavorable for bacterial growth. In this study, we identified bacteria in cultures of different C. albicans isolates from long term, continuous growth by molecular analysis and microscopy. Also, we confirmed the presence of these co-evolving bacteria by identification of S. epidermidis genome segments in sequencing reads of the C. albicans reference strain SC5314 genome sequencing project raw data deposited in GenBank. This result rules out the possibility of laboratory specific contamination. Also, we show that the presence of associated bacteria correlates with antifungal resistance alterations observed in growth under hypoxia. Our findings show the intense interaction between C. albicans yeasts and bacteria and have direct implications in yeast clinical procedures, especially concerning patient treatment.

microbiology↗

bioRxiv2017

Cytokinin signaling alters acid-fast staining of Mycobacterium tuberculosis

It was reported the human-exclusive pathogen Mycobacterium (M.) tuberculosis secretes cytokinins, which previously had only been known as plant hormones. Cytokinins are adenine-based signaling molecules in plants that have never been shown to participate in signal transduction in other kingdoms of life. Here, we show that cytokinins induce the strong expression of the M. tuberculosis gene, Rv0077c. We found that a TetR-like transcriptional regulator, Rv0078, directly repressed expression of the Rv0077c gene. Strikingly, cytokinin-induced expression of Rv0077c resulted in a loss of acid-fast staining of M. tuberculosis. Although acid-fast staining is thought to be associated with changes in the bacterial cell envelope and virulence, Rv0077c-induced loss of acid-fastness did not affect antibiotic susceptibility or attenuate bacterial growth in mice. Collectively, these findings show cytokinins signal transcriptional changes that affect the M. tuberculosis cell envelope, and that cytokinin signaling is no longer limited to the kingdom plantae.

microbiology↗

bioRxiv2017

Determining the Specificity of Cascade Binding, Interference, and Priming in vivo

In CRISPR-Cas immunity systems, short CRISPR RNAs (crRNAs) are bound by CRISPR-associated (Cas) proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to sub-optimal, off-target sites is inert. Our data support a model in which initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5{square} end, whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base-pairing.\n\nIMPORTANCEMany bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61 nt CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base-pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease that destroys the invading DNA molecule and promotes acquisition of new immunity elements. We make the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. By contrast, we show that target degradation and acquisition of new immunity elements requires highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets.

microbiology↗

bioRxiv2017

Gene clusters encoding putative outer membrane electron conduits have specific roles during metal and electrode respiration in Geobacter sulfurreducens

At least five gene clusters in the Geobacter sulfurreducens genome encode putative electron conduits implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and -0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an {Delta}omcBC background, such as extEFG, were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the {Delta}extABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens, depending on the available extracellular electron acceptor.

microbiology↗

bioRxiv2017

Vibrio cholerae genomic diversity within and between patients

Cholera is a severe, waterborne diarrheal disease caused by toxin-producing strains of the bacterium Vibrio cholerae. Comparative genomics has revealed \"waves\" of cholera transmission and evolution, in which clones are successively replaced over decades and centuries. However, the extent of V. cholerae genetic diversity within an epidemic or even within an individual patient is poorly understood. Here, we characterized V. cholerae genomic diversity at a micro-epidemiological level within and between individual patients from Bangladesh and Haiti. To capture within-patient diversity, we isolated multiple (8 to 20) V. cholerae colonies from each of eight patients, sequenced their genomes and identified point mutations and gene gain/loss events. We found limited but detectable diversity at the level of point mutations within hosts (zero to three single nucleotide variants within each patient), and comparatively higher gene content variation within hosts (at least one gain/loss event per patient, and up to 103 events in one patient). Much of the gene content variation appeared to be due to gain and loss of phage and plasmids within the V. cholerae population, with occasional exchanges between V. cholerae and other members of the gut microbiota. We also show that certain intra-host variants have phenotypic consequences. For example, the acquisition of a Bacteroides plasmid and nonsynonymous mutations in a sensor histidine kinase gene both reduced biofilm formation, an important trait for environmental survival. Together, our results show that V. cholerae is measurably evolving within patients, with possible implications for disease outcomes and transmission dynamics.\n\nAuthor SummaryVibrio cholerae is the etiological agent of cholera, a severe diarrheal disease endemic to Bangladesh and responsible for global outbreaks, including one ongoing in Haiti. Certain bacterial pathogens can evolve and diversify within the human host, often altering virulence and antibiotic resistance. However, most examples of within-host evolution have come from chronic infections, in which the pathogen has sufficient time to mutate and diversify, and little attention has been paid to more acute infections such as the one caused by V. cholerae. The goal of this study was to measure the extent of within-host evolution of V. cholerae within individual infected patients. By sequencing multiple bacterial isolates_from each of eight patients from Bangladesh and Haiti, we found that cholera patients can harbor a diverse population of V. cholerae. As expected for an acute infection, this diversity is limited, ranging from zero to three point mutations (single nucleotide variants) per patient. However, gene gain/loss events are more prevalent than point mutations, occurring in every single patient, and sometimes involving the transfer of dozens of genes on plasmids. Even if rare, point mutations and gene gain/loss events may be maintained by natural selection, and can alter clinically-and environmentally-relevant phenotypes such as biofilm formation. Therefore, within-patient evolution has the potential to impact clinical and epidemiological outcomes. Together, our results demonstrate that within-patient evolution may be a general feature of both acute and chronic infections, and that gene gain/loss may be an important but under-appreciated feature of within-host evolution.

microbiology↗

bioRxiv2017

Translational regulation by bacterial small RNAs via an unusual Hfq-dependent mechanism

In bacteria, the canonical mechanism of translational repression by small RNAs (sRNAs) involves sRNA-mRNA base pairing that occludes the ribosome binding site (RBS), directly preventing translation. In this mechanism, the sRNA is the direct regulator, while the RNA chaperone Hfq plays a supporting role by stabilizing the sRNA. There are a few examples where the sRNA does not directly interfere with ribosome binding, yet translation of the target mRNA is still inhibited. Mechanistically, this non-canonical regulation by sRNAs is poorly understood. Our previous work demonstrated repression of the mannose transporter manX mRNA by the sRNA SgrS, but the regulatory mechanism was unknown. Here, we report that manX translation is controlled by a molecular role-reversal mechanism where Hfq, not the sRNA, is the direct repressor. Hfq binding adjacent to the manX RBS is required for sRNA-mediated translational repression. Translation of manX is also regulated by another sRNA, DicF, via the same non-canonical Hfq-dependent mechanism. Our results suggest that the sRNAs recruit Hfq to its binding site or stabilize the mRNA-Hfq complex. This work adds to the growing number of examples of diverse mechanisms of translational regulation by sRNAs in bacteria.

microbiology↗

bioRxiv2017

Mammalian Orthoreovirus Factories Modulate Stress Granule Protein Localization by Interaction with G3BP1

Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2 which promotes formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early, then interferes with SG formation as infection proceeds. In this work, we found that SG associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of SG effector protein, G3BP1, with MRV non-structural protein {sigma}NS, which localizes to VFs via association with VF nucleating protein, NS. Deletion analysis of the {sigma}NS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that the association and VF localization of G3BP1 is not occurring solely through RNA or ribosomal binding, but requires both RNA and ribosomal binding domains of G3BP1 for maximal VFL localization and {sigma}NS association. Co-expression of {sigma}NS and NS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that {sigma}NS association with and relocalization of G3BP1 to the VF periphery plays a role in SG disruption to facilitate MRV replication in the host translational shutoff environment.\n\nIMPORTANCESGs and SG effector proteins have emerged as important, yet poorly understood, players in the hosts innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2 signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of MRV non-structural protein {sigma}NS with major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG associated proteins around the periphery of virus encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.

microbiology↗

bioRxiv2017

The contribution of genetic variation of Streptococcus pneumoniae to the clinical manifestation of invasive pneumococcal disease

BackgroundDifferent clinical manifestations of invasive pneumococcal disease (IPD) have thus far mainly been explained by patient characteristics. Here we studied the contribution of pneumococcal genetic variation to IPD phenotype.\n\nMethodsThe index cohort consisted of 349 patients admitted to two Dutch hospitals between 2000-2011 with pneumococcal bacteraemia. We performed genome-wide association studies to identify pneumococcal lineages, genes and allelic variants associated with 23 clinical IPD phenotypes. The identified associations were validated in a nationwide (n=482) and a post-pneumococcal vaccination cohort (n=121). The contribution of confirmed pneumococcal genotypes to the clinical IPD phenotype, relative to known clinical predictors, was tested by regression analysis.\n\nFindingsThe presence of pneumococcal gene slaA was a nationwide confirmed independent predictor of meningitis (OR=10.5, p=0.001), as was sequence cluster 9 (OR=3.68, p=0.057). A set of 4 pneumococcal genes co-located on a prophage was a confirmed independent predictor of 30-day mortality (OR=3.4, p=0.003). We could detect the pneumococcal variants of concern in these patients blood samples by molecular amplification. In the post-vaccination cohort where the distribution of both patient characteristics and pneumococcal serotypes had changed, the relative importance of the prophage was no longer supported.\n\nInterpretationKnowledge of pneumococcal genotypic variants improved our clinical risk assessment for detrimental manifestations of IPD. This provides us with novel opportunities to target, anticipate or avert the pathogenic effects that are related to particular pneumococcal variants. Therefore, future diagnostics should facilitate prompt appreciation of pathogen diversity in clinical sepsis management. Ongoing surveillance is warranted to monitor the clinical value of information on pathogen variants in dynamic microbial and susceptible host populations.\n\nFundingNone.

microbiology↗

bioRxiv2017

Efficient hydrogen-dependent carbon dioxide reduction by Escherichia coli

The formate hydrogenlyase (FHL) complex of Escherichia coli is normally produced under anaerobic fermentative conditions when its physiological role is to oxidise formate to carbon dioxide and couple that reaction directly to the reduction of protons to molecular hydrogen. This forward reaction is the major route of hydrogen production by E. coli and the physiology, genetics and biochemistry of this process are reasonably well understood. However, studies of the reverse reaction - hydrogen-dependent carbon dioxide reduction - have been rather less extensive. Harnessing the alternative reverse reaction has the potential to unlock FHL as a carbon dioxide cycling enzyme, or could potentially lead to the development of bio-based carbon capture technologies. In this work, it is established that FHL can operate as a highly efficient CO2 reductase. A controllable pressure system was designed with the intention of maximising substrate availability to the FHL enzyme. By placing gaseous CO2 and H2 are under pressure (up to 10 bar), and by using a compartmentalised intact whole cell approach where the produced formate is excreted, the optimised experimental system was observed to convert 100 % of gaseous CO2 to formic acid and generate >500 mM formate in solution at an initial rate of 1.2 g formate per litre per hour.

microbiology↗

bioRxiv2017

The nucleoid as a scaffold for the assembly of bacterial signaling complexes

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.\n\nAUTHOR SUMMARYIn this work, we show that the cytoplasmic chemoreceptor of the Frz chemosensory system, FrzCD, does not bind the cytoplasmic membrane like most MCPs but bind the bacterial nucleoid directly, thus forming distributed protein clusters also containing the Frz kinase. In vitro and in vivo experiments show that DNA-binding is not sequence-specific and is mediated by a basic aminoacid sequence of the FrzCD N-terminal domain. The deletion of this motif abolishes FrzCD DNA-binding and cooperativity in the response to signals. This work shows the importance of the nucleoid in the organization and functioning of cytoplasmic signaling systems in bacteria.

microbiology↗

bioRxiv2017

The global biogeography of amino acid variants within a single SAR11 population is governed by natural selection

The diversity and geographical distribution of populations within major marine microbial lineages are largely governed by temperature and its co-variables. However, neither the mechanisms by which genomic heterogeneity emerges within a single population nor how it drives the partitioning of ecological niches are well understood. Here we took advantage of billions of metagenomic reads to study one of the most abundant and widespread microbial populations in the surface ocean. We characterized its substantial amount of genomic heterogeneity using single-amino acid variants (SAAVs), and identified systematic purifying selection and adaptive mechanisms governing non-synonymous variation within this population. Our Deep Learning analysis of SAAVs across metagenomes revealed two main ecological niches that reflect large-scale oceanic current temperatures, as well as six proteotypes demarcating finer-resolved niches. We identified significantly more protein variants in cold currents and an increased number of protein sweeps in warm currents, exposing a global pattern of alternating genomic diversity for this SAR11 population as it drifts along with surface ocean currents. Overall, the geographic partitioning of SAAVs suggests natural selection, rather than neutral evolution, is the main driver of the evolution of SAR11 in surface oceans.

microbiology↗

bioRxiv2017

Inhibition of NHEJ repair by type II-A CRISPR-Cas systems

CRISPR-Cas systems introduce double strand breaks into DNA of invading genetic material and use DNA fragments to acquire novel spacers during adaptation. Double strand breaks are the substrate of several bacterial DNA repair pathways, paving the way for interactions between them and CRISPR-Cas systems. Here, we hypothesized that non-homologous end joining (NHEJ) interferes with type II CRISPR-Cas systems. We tested this idea by studying the patterns of co-occurrence of the two systems in bacterial genomes. We found that NHEJ and type II-A CRISPR-Cas systems only co-occur once among 5563 fully sequenced prokaryotic genomes. We investigated experimentally the possible molecular interactions causing this negative association using the NHEJ pathway from Bacillus subtilis and the type II-A CRISPR-Cas systems from Streptococcus thermophilus and Streptococcus pyogenes. Our results suggest that the NHEJ system has no effect on type II-A CRISPR-Cas interference and adaptation. On the other hand, we provide evidence for the inhibition of NHEJ repair by the Csn2 protein from type II-A CRISPR-Cas system. Our findings give insights on the complex interactions between CRISPR- Cas systems and repair mechanisms in bacteria and contribute to explain the scattered distribution of CRISPR-Cas systems in bacterial genomes.

microbiology↗

bioRxiv2017

The Epstein-Barr Virus miR-BHRF1 microRNAs Regulate Viral Gene Expression in cis

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant ({triangleup}123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of primiR-BHRF1-2 and 1-3 in the {triangleup}123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the {triangleup}123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis.\n\nHighlightsO_LIThe EBV miR-BHRF1 microRNAs do not up upregulate B cell growth in trans.\nC_LIO_LIEBNA-LP expression is downregulated by pri-miR-BHRF1-2 and 1-3 acting in cis.\nC_LIO_LILoss of miR-BHRF1-2 and 1-3 causes EBNA-LP overexpression and inhibits B cell growth.\nC_LIO_LINovel alternative splicing of EBV Cp/Wp transcripts was identified.\nC_LI

microbiology↗

bioRxiv2017

Human placental syncytiotrophoblasts restrict Toxoplasma gondii vertical transmission at two distinct stages and induce CCL22 in response to infection

Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human mid-gestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at distinct stages of the parasite lytic cycle--at the time of attachment and also during intracellular replication. Utilizing comparative RNAseq transcriptional profiling, we also show that human placental trophoblasts at both mid- and late-stages of gestation induce the chemokine CCL22 in response to T. gondii infection, which relies on the secretion of parasite effector(s). Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy, and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment.\n\nSignificance statementToxoplasma gondii is a major source of congenital disease worldwide and must breach the placental barrier to be transmitted from maternal blood to the developing fetus. The events associated with the vertical transmission of T. gondii are largely unknown. Here, we show that primary human syncytiotrophoblasts, the fetal-derived cells that comprise the primary placental barrier, restrict T. gondii infection at two distinct stages of the parasite life cycle and respond to infection through the induction of the chemokine CCL22. Collectively, our findings provide important insights into the mechanisms by which human syncytiotrophoblasts restrict T. gondii infection at early and late stages of human pregnancy and identify the placental-enriched signaling pathways induced in response to infection.

microbiology↗

bioRxiv2017

The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructure in vitro

The bacterial tubulin FtsZ2 polymerizes to form a discontinuous cytokinetic ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL) and a C-terminal conserved -helix (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The dynamic turnover and treadmilling of clusters of FtsZ protofilaments guides cell wall synthesis and constriction. The nature and regulation of the interactions that result in the assembly of protofilaments into dynamic clusters is unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro. FtsZ lacking its CTL ({Delta}CTL) shows dramatically increased propensity to form long multifilament bundles compared to wildtype (WT). {Delta}CTL has reduced GTP hydrolysis rate compared to WT. However, reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts {Delta}CTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between Z-ring structure and function in bacterial cytokinesis.

microbiology↗

bioRxiv2017

An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e., \"orphan\") operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen P. aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets.

microbiology↗

bioRxiv2017

Detection of live mycobacteria with a solvatochromic trehalose probe for point-of-care tuberculosis diagnosis

AbstractTuberculosis (TB) is the leading cause of death from an infectious bacterial disease. Poor diagnostic tools to detect active disease plague TB control programs and affect patient care. Accurate detection of live Mycobacterium tuberculosis (Mtb), the causative agent of TB, will improve TB diagnosis and patient treatment. We report that live mycobacteria can be specifically detected with a fluorogenic trehalose analog. We designed a 4-N,N-dimethylamino-1,8- naphthalimide-trehalose (DMN-Tre) conjugate that undergoes >700-fold fluorescence increase when transitioned from aqueous to hydrophobic environments. This enhancement occurs upon metabolic conversion of DMN-Tre to trehalose monomycolate and incorporation into the outer membrane. DMN-Tre labeling enabled the rapid, no-wash visualization of mycobacterial and corynebacterial species without nonspecific labeling of Gram-positive or -negative bacteria. DMN-Tre labeling was selective for live mycobacteria and was reduced by treatment with TB drugs. Lastly, DMN-Tre labeled Mtb in TB-positive sputum samples suggesting this operationally simple method may be deployable for TB diagnosis.

microbiology↗

bioRxiv2017

The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135

5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be microbially degraded. However, the physiological, biochemical, and genetic foundation of the microbial catabolism of 5HPA remains unknown. In this study, a gene cluster hpa (which is involved in degradation of 5HPA in Alcaligenes faecalis JQ135) was cloned and HpaM was identified as a novel monocomponent FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) of Pseudomonas putida KT2440. hpaM was heterologously expressed in E. coli BL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography. HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the presence of FAD and NADH, but not of FMN and NADPH. The apparent Km values of HpaM toward 5HPA and NADH were 45.4 and 37.8 , respectively. Results of gene deletion and complementation showed that hpaM was essential for 5HPA degradation in Alcaligenes faecalis JQ135.\n\nImportancePyridine derivatives are ubiquitous in nature and important chemical materials that are currently widely used in agriculture, pharmaceutical, and chemical industries. Thus, the microbial degradation and transformation mechanisms of pyridine derivatives received considerable attention. Decarboxylative hydroxylation was an important degradation process in pyridine derivatives, and previously reported decarboxylative hydroxylations happened in the C3 of the pyridine ring. In this study, we cloned the gene cluster hpa, which is responsible for 5HPA degradation in Alcaligenes faecalis JQ135, thus identifying a novel monocomponent FAD-dependent monooxygenase HpaM. Unlike 3-decarboxylative monooxygenases, HpaM catalyzed decarboxylative hydroxylation in the C2 of the pyridine ring in 5-hydroxypicolinic acid. These findings deepen our understanding of the molecular mechanism of microbial degradation of pyridine derivatives. Furthermore, HpaM offers potential for applications to transform useful pyridine derivatives.

microbiology↗