The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.The symbiosis between legumes and the root nodule bacteria collectively known as rhizobia is of critical agronomic and environmental importance, accounting for the majority of the nitrogen fixed through biological processes. Rhizobia are phylogenetically diverse, falling into five genera of ␣-proteobacteria (Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, and Mesorhizobium) (64, 70) and at least two genera of ␤-proteobacteria (Burkholderia and Ralstonia) (35). It is thought that the rhizobial lineages diverged well before the evolution of legumes and that the genes required for the formation of the symbiosis were subsequently acquired by lateral transfer from undefined sources (8,33). Reflecting the accessory nature of the traits, several species of rhizobia contain the genes required for nodulation and nitrogen fixation on large plasmids that can be cured under laboratory conditions without affecting the survival of the bacteria (31). Exceptions in which the symbiosis genes are encoded on the chromosome include Bradyrhizobium species, in which the symbiosis genes are clustered but not known to be mobile (18), and at least one strain of Mesorhizobium loti, strain ICMP3153, in which the genes are located on a mobile symbiosis island (57).M. loti is the microsymbiont of several Lotus species, including Lotus corniculatus and L. japonicus. The symbiosis island of M. loti strain ICMP3153 was discovered through its ability to transfe...

SummaryGenomics and whole genome sequencing (WGS) have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology.The growth and availability of bench-top WGS analysers has facilitated the feasibility of genomics in clinical and public health microbiology.Given current resource and infrastructure limitations, WGS is most applicable to use in public health laboratories, reference laboratories, and hospital infection control-affiliated laboratories.As WGS represents the pinnacle for strain characterisation and epidemiological analyses, it is likely to replace traditional typing methods, resistance gene detection and other sequence-based investigations (e.g., 16S rDNA PCR) in the near future.Although genomic technologies are rapidly evolving, widespread implementation in clinical and public health microbiology laboratories is limited by the need for effective semi-automated pipelines, standardised quality control and data interpretation, bioinformatics expertise, and infrastructure.

Transformation of a type I SCCmec element into Staphylococcus aureus yielded highly oxacillin-resistant transformants with a reduced growth rate. Faster-growing variants could again be selected at the cost of reduced resistance levels, demonstrating an inverse correlation between oxacillin resistance levels and growth rate.

An association between moenomycin resistance and vancomycin intermediate resistance inStaphylococcus aureus was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn551 insertional mutagenesis of methicillin-resistant S. aureus strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn551 insertions in the fmtC (mprF) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn551 insertion was located in the lysC gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as ␤-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of S. aureus appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.

SummaryGlucosamine-6-P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine-6-P is processed sequentially to UDP-Nacetylglucosamine, while to enter the glycolysis pathway, glucosamine-6-P is isomerized by NagB to fructose-6-P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine-6-P formation from Nacetylglucosamine-6-P, and GlmS in that from fructose-6-P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N -acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N -acetylglucosamine. This and the preferential incorporation of Nacetylglucosamine over glucose into cell wall material showed that N -acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA , nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.