A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.     

Complete Genome Sequence of Staphylococcus aureus Strain JKD6159, a Unique Australian Clone of ST93-IV Community Methicillin-Resistant Staphylococcus aureus

Community methicillin-resistant Staphylococcus aureus (cMRSA) is an emerging issue that has resulted in multiple worldwide epidemics. We report the first complete genome sequence of an ST93-MRSA-IV clinical isolate that caused severe invasive infection and a familial outbreak of skin infection. This isolate is a representative of the most common Australian clone of cMRSA that is more distantly related to the previously sequenced genomes of S. aureus.Staphylococcus aureus is a major cause of both hospital- and community-acquired infections, with rapid emergence of antibiotic resistance, in particular methicillin resistance, adding complexity to the treatment of this organism (3). While previously a hospital problem, methicillin-resistant S. aureus (MRSA) is now being increasingly documented in healthy patients in the community, and these isolates are termed “community MRSA” (cMRSA). A number of cMRSA genomes have been sequenced; however, these are phylogenetically closely related to each other. In contrast, ST93-MRSA-IV, a unique Australian clone, is a singleton by multilocus sequence typing (MLST) eBURST analysis (4). It is now the dominant cMRSA clone in Australia and is associated with both skin infection and severe invasive infection, including necrotizing pneumonia, deep-seated abscesses, and septicemia (5, 10). JKD6159 is a representative ST93-MRSA-IV clinical isolate which caused septicemia and multifocal pulmonary and musculoskeletal abscesses in a previously well intravenous drug user and also resulted in a familial outbreak of skin infection.The genome sequence of S. aureus strain JKD6159 was determined by high-throughput whole-genome shotgun sequencing, using both Illumina GAII (Illumina, CA) and Roche GS FLX Titanium (Roche Diagnostics, Basel, Switzerland) sequencing technologies, producing approximately 164× and 32× coverage of the genome, respectively. The GS FLX Titanium reads were assembled using Newbler 2.0.01.12, resulting in 56 contigs totaling 2.8 Mbp (9). The paired GAII reads were aligned to the contigs using SHRiMP 1.3.2 to identify and correct 74 homopolymeric sequencing errors (11). Optical mapping was used to produce a high-resolution XbaI chromosome restriction map, and the contigs were ordered and oriented against this map using MapSolver 2.1.1 (Opgen). Gap closures were performed by PCR followed by Sanger sequencing of amplification products (3730S genetic analyzer sequencer; Applied Biosystems, CA). The finished sequence was validated by reference to the XbaI optical map, Roche GS FLX Titanium mate pair analysis, and Illumina paired-end-read analysis.Protein coding regions were predicted using GeneMarkS 4.6b, tRNA genes using tRNAscan-SE 1.23, and rRNA genes using RNAmmer 1.2 (2, 7, 8). Gene products were assigned using HMMER 3.0 against the Pfam database (release 23) and BLAST 2.2.23 against RefSeq Proteins (April 2010) and the Conserved Domain Database (v2.22) (1, 6). These automated analyses were followed by manual curation, including comparison with other completed S. aureus genomes.The genome of S. aureus strain JKD6159 consists of a circular 2,811,435-bp chromosome with 33% G+C content—similar to those of other staphylococci—and one circular plasmid of 20,730 bp. A total of 2,605 coding regions, 57 tRNA genes, and 5 rRNA loci were detected. Over 67% of genes were assigned to specific Clusters of Orthologous Groups (COG) Database functional groups, and 40% were assigned an enzyme classification number (12).Initial analysis of the whole-genome sequence of JKD6159 confirms that ST93-MRSA-IV is distantly related to other previously sequenced S. aureus genomes. ST93-MRSA-IV has a distinct accessory genome. There were a number of regions of difference in JKD6159 that contain coding sequences (CDS) not present in any other published S. aureus genomes. Additionally, the ssl gene cluster in JKD6159 appears distinct from other sequenced S. aureus isolates. Comparison with other S. aureus genomes also shows that although JKD6159 carries lukSF-PV (the genes encoding Panton-Valentine leukocidin), there is a relative paucity of virulence factors such as tst-1, genes encoding staphylococcal enterotoxins A to U, and the ACME locus. Further analysis of the genome is now under way to identify factors that might explain the emergence of this MRSA strain in the community.     

Understanding the transmission of Mycobacterium ulcerans: A step towards controlling Buruli ulcer

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rare but chronic debilitating skin and soft tissue disease found predominantly in West Africa and Southeast Australia. While a moderate body of research has examined the distribution of M. ulcerans, the specific route(s) of transmission of this bacterium remain unknown, hindering control efforts. M. ulcerans is considered an environmental pathogen given it is associated with lentic ecosystems and human-to-human spread is negligible. However, the pathogen is also carried by various mammals and invertebrates, which may serve as key reservoirs and mechanical vectors, respectively. Here, we examine and review recent evidence from these endemic regions on potential transmission pathways, noting differences in findings between Africa and Australia, and summarising the risk and protective factors associated with Buruli ulcer transmission. We also discuss evidence suggesting that environmental disturbance and human population changes precede outbreaks. We note five key research priorities, including adoption of One Health frameworks, to resolve transmission pathways and inform control strategies to reduce the spread of Buruli ulcer.     

The Lipoprotein LpqW Is Essential for the Mannosylation of Periplasmic Glycolipids in Corynebacteria

Phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) are essential components of the cell wall and plasma membrane of mycobacteria, including the human pathogen Mycobacterium tuberculosis, as well as the related Corynebacterineae. We have previously shown that the lipoprotein, LpqW, regulates PIM and LM/LAM biosynthesis in mycobacteria. Here, we provide direct evidence that LpqW regulates the activity of key mannosyltransferases in the periplasmic leaflet of the cell membrane. Inactivation of the Corynebacterium glutamicum lpqW ortholog, NCgl1054, resulted in a slow growth phenotype and a global defect in lipoglycan biosynthesis. The NCgl1054 mutant lacked LAMs and was defective in the elongation of the major PIM species, AcPIM2, as well as a second glycolipid, termed Gl-X (mannose-α1–4-glucuronic acid-α1-diacylglycerol), which function as membrane anchors for LM-A and LM-B, respectively. Elongation of AcPIM2 and Gl-X was found to be dependent on expression of polyprenol phosphomannose (ppMan) synthase. However, the ΔNCgl1054 mutant synthesized normal levels of ppMan, indicating that LpqW is not required for synthesis of this donor. A spontaneous suppressor strain was isolated in which lipoglycan synthesis in the ΔNCgl1054 mutant was partially restored. Genome-wide sequencing indicated that a single amino acid substitution within the ppMan-dependent mannosyltransferase MptB could bypass the need for LpqW. Further evidence of an interaction is provided by the observation that MptB activity in cell-free extracts was significantly reduced in the absence of LpqW. Collectively, our results suggest that LpqW may directly activate MptB, highlighting the role of lipoproteins in regulating key cell wall biosynthetic pathways in these bacteria.     

Comparative analysis of the first complete Enterococcus faecium genome

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infections in health care facilities around the globe. In particular, infections caused by vancomycin-resistant Enterococcus faecium are becoming increasingly common. Comparative and functional genomic studies of E. faecium isolates have so far been limited owing to the lack of a fully assembled E. faecium genome sequence. Here we address this issue and report the complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998. The genome comprises a 2.9-Mb circular chromosome and three circular plasmids. The chromosome harbors putative E. faecium virulence factors such as enterococcal surface protein, hemolysin, and collagen-binding adhesin. Aus0004 has a very large accessory genome (38%) that includes three prophage and two genomic islands absent among 22 other E. faecium genomes. One of the prophage was present as inverted 50-kb repeats that appear to have facilitated a 683-kb chromosomal inversion across the replication terminus, resulting in a striking replichore imbalance. Other distinctive features include 76 insertion sequence elements and a single chromosomal copy of Tn1549 containing the vanB vancomycin resistance element. A complete E. faecium genome will be a useful resource to assist our understanding of this emerging nosocomial pathogen.