The C-terminal domain of feline and bovine SAMHD1 proteins has a crucial role in lentiviral restriction

SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.

Distinct genetic profiles of Leishmania (Viannia) braziliensis associate with clinical variations in cutaneous-leishmaniasis patients from an endemic area in Brazil

American tegumentary leishmaniasis (ATL) samples obtained from the lesions of patients with typical (n = 25, 29%), atypical (n = 60, 69%) or both (n = 2%) clinical manifestations were analysed by multilocus enzyme electrophoresis, hsp70 restriction-fragment length polymorphism (PCR-RFLP), hsp70 sequencing and phylogenetics methods. The hsp70 PCR-RFLP analysis revealed two different profiles whose the most samples differed from those expected for Leishmania braziliensis and the other Leishmania species tested: of 39 samples evaluated, two (5%) had a restriction profile corresponding to L. braziliensis, and 37 (95%) had a restriction profile corresponding to a variant pattern. A 1300-bp hsp70 gene fragment was sequenced to aid in parasite identification and a phylogenetic analysis was performed including 26 consensus sequences from the ATL patient's samples and comparing to other Leishmania and trypanosomatids species. The dendrogram allowed to observe a potential population structure of L. braziliensis complex in the studied region, emphasizing that the majority of clinical samples presented a variant genetic profile. Of interest, the L. braziliensis diversity was associated with different clinical manifestations whose parasites with hsp70 variant profile were associated with atypical lesions. The results may be helpful to improve the diagnosis, treatment and control measures of the ATL in endemic areas.

Hybridization Processes in Putative Hybrid Swarms of Scots Pine and Mountain Dwarf Pine as Revealed by Chloroplast DNA

Gene flow among individual trees of Pinus sylvestris and P. mugo putative hybrid swarms in Slovakia was fol-lowed at four localities using the species-diagnostic cpDNA trnV-trnH/Hinf I restriction profile. Variable proportions of P. sylvestris and P. mugo haplotypes were revealed among the sampled localities. Low between-habitus consistency of the trees and their cpDNA haplotypes indicates the hybrid nature of the swarms. Molecular analy-sis based on mutual comparison of the haplotypes of a given tree and its embryos suggests direct and recipro-cal hybridization between trees of the P. sylvestris and P. mugo haplotypes. Besides conspecific embryos resulting from hybridization of trees with the same haplotype (P. sylvestris × P. sylvestris and P. mugo × P. mugo), hybrid embryos of P. sylvestris × P. mugo (8.03%) and P. mugo × P. sylvestris (11.50%) were also detected in open-pollinated offspring. The results are discussed from the standpoint of primary and introgressive hybridization between the parental species.

Selection and Characterization of a Multivalent Salmonella Phage and Its Production in a Nonpathogenic Escherichia coli Strain

ABSTRACT We report the selection and amplification of the broad-host-range Salmonella phage phi PVP-SE1 in an alternative nonpathogenic host. The lytic spectrum and the phage DNA restriction profile were not modified upon replication in Escherichia coli Bl21, suggesting the possibility of producing this phage in a nonpathogenic host, contributing to the safety and easier approval of a product based on this Salmonella biocontrol agent.

CAPS Analysis: A Possible Tool to Detect and Group Geminiviruses Infecting Some Fibre Crops and Weeds

Some fibre crops and weeds, showing typical symptoms caused by begomoviruses, were subjected to PCR amplification using different sets of primers specific for DNA A and DNA β molecules of begomoviruses. Restriction digestion of full length DNA A and DNA β using different enzymes revealed variation in restriction profiles amongst the virus isolates in the present study. Moreover, variation in the restriction profile of different DNA β molecule with that of their corresponding reported sequences were also noticed. Cleaved amplified polymorphic sequence (CAPS) analysis of DNA β revealed little or no genetic distance between the begomoviruses infecting Sida acuminata and S. rhombifolia and also among the begomoviruses infecting Urena, Hibiscus cannabinus, and H. sabdariffa. But the same analysis with full length DNA A showed close phylogenetic relationships between begomoviruses infecting H. cannabinus and H. sabdariffa. Hence, for wide spread variation and unavailability of any sequence information CAP analysis might be a useful tool to detect and group begomoviruses. Accepted for publication 9 October 2008. Published 2 February 2009.

Vancomycin-Resistant Staphylococcus aureus Isolates Associated with Inc18-Like vanA Plasmids in Michigan

ABSTRACT Five of the seven cases of vancomycin-resistant Staphylococcus aureus (VRSA) infection identified to date have occurred in southeastern Michigan. VRSA isolates from the four most recent cases (all from Michigan) were characterized. The vanA gene was localized to a single plasmid in each VRSA isolate. The pulsed-field gel electrophoresis patterns of chromosomal DNA and the restriction profile of the plasmid demonstrated that the four isolates were unique and differed from the first three VRSA isolates. Vancomycin-resistant Enterococcus (VRE) isolates, all of which were Enterococcus faecalis, were recovered from case patients 4 to 6. Each VRE isolate transferred vancomycin resistance to E. faecalis JH2-2 by conjugation. PCRs for vanA and the Inc18-like plasmid genes traA and repR confirmed the presence of an Inc18-like vanA plasmid in all VRE isolates and transconjugants. An Inc18-like vanA plasmid was identified in the VRSA isolate from case patient 7. These findings suggest a role of Inc18-like plasmids as vanA donors.

Genetic and phenotypic diversity of Bifidobacterium thermacidophilum fecal isolates from newborns

This study was undertaken to genetically identify and phenotypically characterize 14 bifidobacteria isolated from 20 breast-fed newborns. These isolates showed 98%–99% similarity to Bifidobacterium thermacidophilum subsp. suis based on 16S rDNA. Further analysis by pulsed-field gel electrophoresis of chromosomal DNA digested with XbaI revealed 4 distinct restriction patterns. The predominant pattern, shared by 8 (57%) isolates, produced a macro-restriction profile with about 13 large fragments ranging in size from >242.5 to 23.1 kb, whereas the other 6 displayed 3 distinct restriction profiles all characterized by more micro- than macro-restriction, with fragments ranging in size from 97 to 9.4 kb. Phenotypic characteristics, including carbohydrate fermentation profile, maximal growth temperature, and antibiotic susceptibility, varied widely even among strains showing the same restriction profile. The presence of B. thermacidophilum in stools of newborn infants may indicate the potential of these bacteria for aiding the development of the intestinal ecosystem.

Morphological and Genetic Diversity of Temperate Phages in Clostridium difficile

ABSTRACT Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages φC2, φC5, and φCD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.

Spread of Staphylococcus aureus resistant to penicillin and tetracycline within and between dairy herds

One hundred and seven bovine isolates of penicillin and tetracycline resistant Staphylococcus aureus, recovered from 25 different dairy herds in various parts of Norway, were characterized using antimicrobial susceptibility testing, multilocus enzyme electrophoresis, ribotyping, plasmid analysis and serotyping of capsular polysaccharide. Forty-one isolates from one particular herd, 37 isolates from 5 herds that used a common pasture and milking parlour in summer and 21 isolates from 12 herds in 8 different counties belonged to the same strain. The remaining 8 isolates, which originated from herds in 5 different counties, were assigned to 6 different strains. Seven out of these 8 isolates had the same plasmid restriction profile. In conclusion, penicillin and tetracycline resistant S. aureus occurring in dairy herds in Norway mainly seems to represent one particular strain that has achieved widespread distribution or belong to one of several different strains carrying identical plasmids.