Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291.

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

Phylogenetic characterization and novelty of organic sulphur metabolizing genes of Rhodococcus spp. (Eu-32).

Rhodococcus spp. (Eu-32) has the unique ability to metabolize organic sulphur containing compounds like dibenzothiophene through an extended sulphur specific pathway (Akhtar et al., in FEMS Microbiol Lett 301:95-102, 2009). Efforts were made to isolate and characterize the presumed desulphurizing genes (dszABC) involved in the sulphur specific pathway of isolate Eu-32 by employing standard and degenerate polymerase chain reaction primers. The partial dszA gene sequence of isolate Eu-32 showed 92% sequence identity with a putative FMNH-2 dependent monooxygenase of Rhodococcus erythropolis PR4. The dszC gene sequence showed 99% homology with the dibenzothiophene monooxygenase desulphurizing enzyme of another Rhodococcus species. The dszB gene was not unambiguously identified. A phylogenetic analysis by maximum likelihood method of the 16S rRNA gene and deduced DszA and C amino acid sequences suggest that horizontal gene transfer events might have taken place during the evolution of desulphurizing genes of Rhodococcus spp. (Eu-32).

Bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong, China.

We investigated the bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong in the Yunnan Province, China, using direct molecular analyses. The Langpu (LP) laminated mat was found by the side of a boiling pool with temperature of 60-65 °C and a pH of 8.5, while the Tengchong (TC) streamer mat consisted of white streamers in a slightly acidic (pH 6.5) hot pool outflow with a temperature of 72 °C. Four 16S rRNA gene clone libraries were constructed and restriction enzyme analysis of the inserts was used to identify unique sequences and clone frequencies. From almost 200 clones screened, 55 unique sequences were retrieved. Phylogenetic analysis showed that the LP mat consisted of a diverse bacterial population [Cyanobacteria, Chloroflexi, Chlorobia, Nitrospirae, 'Deinococcus-Thermus', Proteobacteria (alpha, beta and delta subdivisions), Firmicutes, Bacteroidetes and Actinobacteria], while the archaeal population was dominated by methanogenic Euryarchaeota and Crenarchaeota. In contrast, the TC streamer mat consisted of a bacterial population dominated by Aquificae, while the archaeal population also contained Korarchaeota as well as Crenarchaeota and methanogenic Euryarchaeota. These mats harboured clone sequences affiliated to unidentified lineages, suggesting that they are a potential source for discovering novel bacteria and archaea.

Phages in nature.

Bacteriophages or phages are the most abundant organisms in the biosphere and they are a ubiquitous feature of prokaryotic existence. A bacteriophage is a virus which infects a bacterium. Archaea are also infected by viruses, whether these should be referred to as 'phages' is debatable, but they are included as such in the scope this article. Phages have been of interest to scientists as tools to understand fundamental molecular biology, as vectors of horizontal gene transfer and drivers of bacterial evolution, as sources of diagnostic and genetic tools and as novel therapeutic agents. Unraveling the biology of phages and their relationship with their hosts is key to understanding microbial systems and their exploitation. In this article we describe the roles of phages in different host systems and show how modeling, microscopy, isolation, genomic and metagenomic based approaches have come together to provide unparalleled insights into these small but vital constituents of the microbial world.

Metagenomics and recovery of enzyme genes from alkaline saline environments.

Enzymes functioning at alkaline pH are widely used in the detergent industry as additives to improve the stain removal properties of domestic and industrial cleaning products. This industry provides by far the major mass market for enzymes. With constantly changing formulations in detergents and concerns over energy demands, new and improved enzymes are constantly in demand. Soda lakes host dense populations of alkali-loving microbes and, as such, provide vast reservoirs of potentially useful enzymes for such an industry. Traditional recovery methods for new enzymes have involved the isolation of microbes, preferably from a compatible chemical environment such as a soda lake, followed by screening of the isolates for useful enzymic activity. At least two commercially significant enzymes originating from soda lake microbes have been marketed following this route. However, the failure to cultivate more than a small percentage of microbes from most environments necessarily markedly reduces the recovery of new enzymes. In recent years, interest has focussed on more comprehensive recovery methods based around detecting appropriate enzyme genes in nucleic acids extracted from potentially useful sites, thus maximizing coverage of the whole genetic resource in a particular biotope. Here we review progress to date in soda lake biotopes and discuss ways the field may develop in the future.

Microbial biogeography of six salt lakes in Inner Mongolia, China, and a salt lake in Argentina.

We used cultivation-independent methods to investigate the prokaryotic biogeography of the water column in six salt lakes in Inner Mongolia, China, and a salt lake in Argentina. These lakes had different salt compositions and pH values and were at variable geographic distances, on both local and intercontinental scales, which allowed us to explore the microbial community composition within the context of both contemporary environmental conditions and geographic distance. Fourteen 16S rRNA gene clone libraries were constructed, and over 200 16S rRNA gene sequences were obtained. These sequences were used to construct biotic similarity matrices, which were used in combination with environmental similarity matrices and a distance matrix in the Mantel test to discover which factors significantly influenced biotic similarity. We showed that archaeal biogeography was influenced by contemporary environmental factors alone (Na+, CO3(2-), and HCO3(-) ion concentrations; pH; and temperature). Bacterial biogeography was influenced both by contemporary environmental factors (Na+, Mg2+, and HCO3(-) ion concentrations and pH) and by geographic distance.

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments.

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.

Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China.

BACKGROUND: We are profoundly ignorant about the diversity of viruses that infect the domain Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus. RESULTS: Haloarchaeal strains with 16S rRNA gene sequences 98% identical to Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content ofapproximately 65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained. CONCLUSION: The virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system.

Caldalkalibacillus thermarum gen. nov., sp. nov., a novel alkalithermophilic bacterium from a hot spring in China.

A thermophilic, alkaliphilic and catalase-positive bacterium, designated strain HA6(T), was isolated from a hot spring in China. The strain was aerobic and chemo-organotrophic and grew optimally at 60 degrees C, pH 8.5 and 1.5 % (w/v) NaCl. The cells were Gram-positive rods, forming single terminal endospores. The predominant cellular fatty acids were iso-C(15 : 0) and iso-C(17 : 0). The cell-wall peptidoglycan contained meso-diaminopimelic acid. The genomic DNA G+C content was 45.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain HA6(T) formed a distinct lineage within the family Bacillaceae and was most closely related to Bacillus horti K13(T) and Bacillus smithii DSM 4216(T), with sequence similarities of 91.8 and 93.1 %, respectively. On the basis of its physiological and molecular properties, strain HA6(T) should be placed in a novel genus and species, for which the name Caldalkalibacillus thermarum gen. nov., sp. nov. is proposed. The type strain of Caldalkalibacillus thermarum is strain HA6(T) (=CGMCC 1.4242(T)=JCM 13486(T)).

Phylogenetic analysis of bacterial isolates from man-made high-pH, high-salt environments and identification of gene-cassette-associated open reading frames.

Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette-specific primers. Different gene-cassette-linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated.

Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples.

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

Analysis of the virus population present in equine faeces indicates the presence of hundreds of uncharacterized virus genomes.

Virus DNA was isolated from horse faeces and cloned in a sequence-independent fashion. 268 clones were sequenced and 178140 nucleotides of sequence obtained. Statistical analysis suggests the library contains 17560 distinct clones derived from up to 233 different virus genomes. TBLASTX analysis showed that 32% of the clones had significant identity to GenBank entries. Of these 63% were viral; 20% bacterial; 7% archaeal; 6% eukarya; and 5% were related to mobile genetic elements. Fifty-two percent of the virus identities were with Siphoviridae; 26% unclassified phages; 17% Myoviridae; 4% Podoviridae; and one clone (2%) was a vertebrate Orthopoxvirus. Genes coding for predicted virus structural proteins, proteases, glycosidases and nucleic acid-binding proteins were common.

A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures.

Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000. Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis. Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga-Flexibacter-Bacteroides group. Similar clones were identified in the SS, but Bacillus spp. predominated. Many of the clones were most closely related to organisms already identified in alkaline or saline environments. Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample. A novel cellulase activity was identified and characterized in each. The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris. The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods.

DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/ Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/ Aeromonas/Vibrio part of the gamma3 subdivision of the Proteobacteria.

Phylogenetic analysis of different isolates of Sulfobacillus spp. isolated from uranium-rich environments and recovery of genes using integron-specific primers.

The isolation and phylogenetic characterization of acidophilic moderate thermophilic bacteria from different locations of uranium mines and a uranium processing mill in Pakistan is reported. The dominant culturable bacteria found were related to Sulfobacillus thermosulfidooxidans in all the samples analyzed. Different strains displayed different levels of identity (95-97%) to 16S rDNA of known strains of this species, indicating group heterogeneity. Genomic DNA from five isolates was subjected to amplification using integron-specific primers HS286 and HS287. Recovery of different integron-linked genes from one of the isolates indicated the usefulness of this approach for gene mining in place of traditional gene recovery methodologies.

Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries.

A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.