Characterizing the microbial metagenome of calcareous stromatolite formations in the San Felipe Creek in the Anza Borrego Desert.

We describe the metagenome composition, community functional annotation, and prokaryote diversity in calcareous stromatolites from a dry stream bed of the San Felipe Creek in the Anza Borrego Desert. Analyses show a community capable of nitrogen fixation, assimilatory nitrate reduction, biofilm formation, quorum sensing, and potential thick-walled akinete formation for desiccation resistance.

Polynucleotide phosphorylase plays an important role in the generation of spontaneous mutations in Escherichia coli.

Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rif(r) and the gyrB gene leading to Nal(r) and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation.

The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

Engineering an incubation environment that mimics in situ conditions for in vitro coastal microbiome studies.

Coastal environments are dynamic and can vary widely on short- or long-term scales depending on location and weather. Incubation equipment that reflects these changes through programmable gradient light and temperature cycles would permit more precise in vitro coastal microbiome studies. Here we present an open-source incubation environment that mimics in situ conditions for in vitro coastal microbiome studies using a modified shaking water bath that has fully customizable temperature and light gradients that can also mimic real-time field conditions. We compared coastal microbial community profiles incubated in situ and in our build mimicking field conditions over 48 h. Analyses of congruence indicated significant overlap (p > 0.2) between microbial communities incubated in situ and in vitro at each time point.

Selective advantage of resistant strains at trace levels of antibiotics: a simple and ultrasensitive color test for detection of antibiotics and genotoxic agents.

Many studies have examined the evolution of bacterial mutants that are resistant to specific antibiotics, and many of these focus on concentrations at and above the MIC. Here we ask for the minimum concentration at which existing resistant mutants can outgrow sensitive wild-type strains in competition experiments at antibiotic levels significantly below the MIC, and we define a minimum selective concentration (MSC) in Escherichia coli for two antibiotics, which is near 1/5 of the MIC for ciprofloxacin and 1/20 of the MIC for tetracycline. Because of the prevalence of resistant mutants already in the human microbiome, allowable levels of antibiotics to which we are exposed should be below the MSC. Since this concentration often corresponds to low or trace levels of antibiotics, it is helpful to have simple tests to detect such trace levels. We describe a simple ultrasensitive test for detecting the presence of antibiotics and genotoxic agents. The test is based on the use of chromogenic proteins as color markers and the use of single and multiple mutants of Escherichia coli that have greatly increased sensitivity to either a wide range of antibiotics or specific antibiotics, antibiotic families, and genotoxic agents. This test can detect ciprofloxacin at 1/75 of the MIC.

Antibiotic sensitivity profiles determined with an Escherichia coli gene knockout collection: generating an antibiotic bar code.

We have defined a sensitivity profile for 22 antibiotics by extending previous work testing the entire KEIO collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to 1 of 14 different antibiotics (ciprofloxacin, rifampin [rifampicin], vancomycin, ampicillin, sulfamethoxazole, gentamicin, metronidazole, streptomycin, fusidic acid, tetracycline, chloramphenicol, nitrofurantoin, erythromycin, and triclosan). We screened one or more subinhibitory concentrations of each antibiotic, generating more than 80,000 data points and allowing a reduction of the entire collection to a set of 283 strains that display significantly increased sensitivity to at least one of the antibiotics. We used this reduced set of strains to determine a profile for eight additional antibiotics (spectinomycin, cephradine, aztreonem, colistin, neomycin, enoxacin, tobramycin, and cefoxitin). The profiles for the 22 antibiotics represent a growing catalog of sensitivity fingerprints that can be separated into two components, multidrug-resistant mutants and those mutants that confer relatively specific sensitivity to the antibiotic or type of antibiotic tested. The latter group can be represented by a set of 20 to 60 strains that can be used for the rapid typing of antibiotics by generating a virtual bar code readout of the specific sensitivities. Taken together, these data reveal the complexity of intrinsic resistance and provide additional targets for the design of codrugs (or combinations of drugs) that potentiate existing antibiotics.

Draft Genome Sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, Bacillus sp. Strain V59.32b, Bacillus sp. Strain MER_TA_151, and Paenibacillus sp. Strain MER_111, Isolated from Cleanrooms Where the Viking and Mars Exploration Rover Spacecraft Were Assembled.

We report the draft genome sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, and Bacillus sp. strain V59.32b, isolated from the Viking spacecraft assembly cleanroom, and Bacillus sp. strain MER_TA_151 and Paenibacillus sp. strain MER_111, isolated from the Mars Exploration Rover (MER) assembly cleanroom.

Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of "codrugs" that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's "intrinsic resistome," which provides innate resistance to antibiotics.

Extreme dNTP pool changes and hypermutability in dcd ndk strains.

Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of Escherichia coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.

Identification of DNA Methylation-Independent Epigenetic Events Underlying Clear Cell Renal Cell Carcinoma.

Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation-independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954-64. ©2016 AACR.

Determination of hypersensitivity to genotoxic agents among Escherichia coli single gene knockout mutants.

We have tested the KEIO collection of 3985 different viable single gene knockouts in Escherichia coli to identify genes whose loss increases sensitivity to one or more of six different chemotherapeutic agents and mutagens: Bleomycin (BLM), Cisplatin (CPT), ICR-191 (ICR), 5-azacytidine (5AZ), Zebularine (ZEB), and 5-bromo-2'-deoxyuridine (5BdU). We discovered a set of 156 strains that display a significant increase in sensitivity to at least one of the agents tested. Each genotoxic agent generates a distinct "sensitivity profile" that is characteristic of the agent. Comparison with an independent study of sensitivity profiles for an extensive set of antibiotics pinpoints those effects that are relatively specific for each agent. In some cases engineered double mutants have greatly increased effects. These results provide insight into the mechanism of action of each agent, and define targets for the design of co-drugs that can potentiate these agents. An example is the finding that mutants lacking one of several genes in the folate biosynthetic pathway are hypersensitive to ZEB, leading to a demonstration of synergy between trimethoprim and ZEB.