Community Reserves: Their significance for the conservation of mammals in a mosaic of community-managed lands in Meghalaya, Northeast India.

Community Reserves (CRs) have been advocated for increasing the protected area coverage in northeast India where the land is primarily owned and managed by local indigenous institutions. To understand the significance of these reserves for the conservation of mammals, we investigated the diversity and abundance of mammals in five CRs in the Khasi Hills of Meghalaya as well as interviewed 75 local villagers to assess the hunting practices and perceptions of the Indigenous Khasis on mammals. We employed 60 camera traps in the CRs and undertook a recce survey (day-time and night-time) for capturing the diversity in the CRs. We used photo-capture rate and encounter rate as indices of relative abundance in the CRs. We used an exact multinomial test to test differences of opinion among the respondents of the five CRs. We found a relatively low abundance of mammals in the CRs, yet they persist. A total of 28 species were detected through camera trapping and recce survey and an additional 12 species were reported by respondents to also occur in the CRs. Among the respondents, it was believed that the decline in mammal populations was largely driven by habitat loss and degradation (82.67%) while only a few believed it was also driven by hunting (5.33%). Respondents also believed that the presence of the reserves and awareness programs taken under them had also led to a reduction in hunting (20%) in their area. Although, some attributed it to a general decline in wildlife populations and forest cover (21.33%). Thus, despite these CRs being small (<2 km2) and isolated, they still harbour mammal species and are important for retaining remnant forest patches in a landscape that is highly fragmented.

A small derivative of the broad-host-range plasmid RK2 which can be switched from a replicating to a non-replicating state as a response to an externally added inducer.

TrfA is the only plasmid-encoded protein required for RK2 replication. We report here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting construct, pJBSD1, was found to replicate in Escherichia coli DH5a (recA(-)) only in the presence of a Pm inducer. In two tested E. coli recA(+) strains pJBSD1 could replicate in the absence of inducer, but a replication inducer-dependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be used as a conditional suicide vector system for targeted chromosomal integration via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology.

Genetic antagonism and hypermutability in Mycobacterium smegmatis.

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.

The host range of RK2 minimal replicon copy-up mutants is limited by species-specific differences in the maximum tolerable copy number.

The minimal replicon of the broad-host-range plasmid RK2 consists of a gene, trfA (trans-acting replication), encoding a protein required for initiation of plasmid replication. The TrfA protein binds to iterons in the cis-acting origin of vegetative replication (oriV), but the exact mechanism by which TrfA-mediated replication initiation takes place is not known. We report here the isolation and characterization of five mini RK2 trfA mutant plasmids with an elevated plasmid copy number, four in Pseudomonas aeruginosa and one in Azotobacter vinelandii. The mutations are localized between or downstream of previously reported Escherichia coli copy-up mutations in trfA, and one of the mutations has been described earlier as an independent copy-up isolate in E. coli. The five mutant plasmids were all moderately copy up in both E. coli and their host of origin, in spite of the use of isolation procedures which were expected to select efficiently in favor of plasmid mutants specifying high copy numbers. In contrast, previously described high copy-up mutants isolated in E. coli could not be established in P. aeruginosa and A. vinelandii. These high copy-up mutants were shown to induce cell killing in E. coli under conditions where the plasmid copy number was increased as a physiological response to reduced growth rate. We propose that the reason for this killing effect is that the copy number under these conditions exceeds an upper tolerance level specific for E. coli. By assuming that the corresponding tolerance level is lower in P. aeruginosa and A. vinelandii than in E. coli, and that the mechanism of copy number regulation is similar, the model can explain the phenotypes of all tested copy up mutants in these two hosts. Analogous studies were also performed in Salmonella typhimurium and Acetobacter xylinum. The data obtained in these studies indicate that the above model is probably generally true for gram-negative bacteria, and the results also indicate that the maximum tolerable copy number is surprisingly low in some hosts.

The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations.

The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein.

Species-dependent phenotypes of replication-temperature-sensitive trfA mutants of plasmid RK2: a codon-neutral base substitution stimulates temperature sensitivity by leading to reduced levels of trfA expression.

TrfA is the only plasmid-encoded protein required for initiation of replication of the broad-host-range plasmid RK2. Here we describe the isolation of four trfA mutants temperature sensitive for replication in Pseudomonas aeruginosa. One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been previously described to be temperature sensitive for replication, but poorly functional, in Escherichia coli. The remaining three mutants were identical, and each of them carried two mutations, one leading to substitution of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral mutation changing the codon for glycine 235 from GGC to GGU (mutation 235). Neither of the two mutations caused a temperature-sensitive phenotype alone in P. aeruginosa, and the effect of the neutral mutation was caused by its ability to strongly reduce the trfA expression level. The double mutant and mutant 163C could not be stably maintained in E. coli, but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced the trfA expression level also in E. coli. The glycine 85 codon in trfA mRNA is GGU, and a change of this to GGC did not significantly affect expression. In addition, we found that wild-type trfA was expressed at much lower levels in E. coli than in P. aeruginosa, indicating that this level is a key parameter in the determination of the temperature-sensitive phenotypes in different species. The E. coli lacZ gene was translationally fused at the 3' end and internally in trfA, in both cases leading to elimination of the effect of mutation 235 on expression. We therefore propose that this mutation acts through an effect on mRNA structure or stability.

Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria.

This report describes the construction and use of improved broad-host-range expression vectors based on the previously constructed pJB137 and pJB653 plasmids (Blatny et al., 1997). These vectors contain the minimal replicon of RK2 and the inducible Pu or Pm promoters together with their regulatory xylR or xylS genes, respectively, from the Pseudomonas putida TOL plasmid pWWO. A set of ATG vectors were derived from pJB653, and these vectors are characterized by the relatively small size, the presence of multiple cloning sites downstream of Pm, the establishment of their nucleotide sequence, the presence of RK2 oriT, and different antibiotic selection markers. The copy numbers of all the vectors can easily be modified by using copy-up mutations of the trfA gene, required for initiation of replication of RK2 replicons. The vectors were used to study the expression levels of the Acetobacter xylinum phosphoglucomutase gene celB and the two commonly used reporter genes luc and cat in Escherichia coli, Pseudomonas aeruginosa, and Xanthomonas campestris. Good induction properties and tight regulation of Pm were achieved in all three species tested, and higher gene expression levels were obtained by using the ATG vectors compared to pJB653. By introducing different trfA copy-up mutations into the vectors, a wide range of gene expression levels from Pu and Pm were obtained in E. coli. Induced expression levels of luc, cat, and celB from Pm were found to be comparable to or higher than those from the Ptrc and PT7 promoters located on high copy number plasmids. The induced levels of Luc activity were higher in P. aeruginosa than in E. coli, indicating that these vectors may be useful for maximization of gene expression in strains other than E. coli. We believe that the well-characterized vectors described here are useful for gene expression studies and routine cloning experiments in many Gram-negative bacteria.