Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Characterization of the MSMEG_2631 gene (mmp) encoding a multidrug and toxic compound extrusion (MATE) family protein in Mycobacterium smegmatis and exploration of its polyspecific nature using biolog phenotype microarray.

In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc(2)155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na(+)-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions.

Albinism and disease causing pathogens in Tanzania: are alleles that are associated with OCA2 being maintained by balancing selection?

Oculocutaneous albinism type 2 (OCA2) is present at significantly higher frequencies in sub-Saharan African populations compared to populations in other regions of the world. In Tanzania and other sub-Saharan countries, most OCA2 is associated with a common 2.7kb deletion allele. Leprosy is also in high prevalence in sub-Saharan African populations. The infectious agent of leprosy, Mycobacterium leprae, contains a gene, 38L, that is similar to OCA2. Hypopigmented patches of skin are early symptoms that present with infection of leprosy. In consideration of both the genetic similarity of OCA2 and the 38L gene of M. leprae and the involvement of pigmentation in both disorders, we hypothesized that the high rates of OCA2 may be due to heterozygote advantage. Hence, we hypothesized that carriers of the 2.7kb deletion allele of OCA2 may provide a protective advantage from infection with leprosy. We tested this hypothesis by determining the carrier frequency of the 2.7kb deletion allele from a sample of 240 individuals with leprosy from Tanzania. The results were inconclusive due to the small sample size; however, they enabled us to rule out a large protective effect, but perhaps not a small advantage. Mycobacterium tuberculosis is another infectious organism prevalent in sub-Saharan Africa that contains a gene, arsenic-transport integral membrane protein that is also similar to OCA2. Interestingly, chromosomal region 15q11-13, which also contains OCA2, was reported to be linked to tuberculosis susceptibility. Although variants within OCA2 were tested for association, the 2.7kb deletion allele of OCA2 was not tested. This led us to hypothesize that the deletion allele may confer resistance to susceptibility. Confirmation of our hypothesis would enable development of novel pharmocogenetic therapies for the treatment of tuberculosis, which in turn, may enable development of drugs that target other pathogens that utilize a similar infection mechanism as M. tuberculosis. From an evolutionary perspective, confirmation of our hypothesis may provide another example of heterozygote advantage.

Mycobacterium marinum SecA2 promotes stable granulomas and induces tumor necrosis factor alpha in vivo.

SecA2 is an ATPase present in some pathogenic Gram-positive bacteria, is required for translocation of a limited set of proteins across the cytosolic membrane, and plays an important role in virulence in several bacteria, including mycobacteria that cause diseases such as tuberculosis and leprosy. However, the mechanisms by which SecA2 affects virulence are incompletely understood. To investigate whether SecA2 modulates host immune responses in vivo, we studied Mycobacterium marinum infection in two different hosts: an established zebrafish model and a recently described mouse model. Here we show that M. marinum ΔsecA2 was attenuated for virulence in both host species and SecA2 was needed for normal granuloma numbers and for optimal tumor necrosis factor alpha response in both zebrafish and mice. M. marinum ΔsecA2 was more sensitive to SDS and had unique protrusions from its cell envelope when examined by cryo-electron tomography, suggesting that SecA2 is important for bacterial cell wall integrity. These results provide evidence that SecA2 induces granulomas and is required for bacterial modulation of the host response because it affects the mycobacterial cell envelope.

Identification of a glucose permease from Mycobacterium smegmatis mc2 155.

We report here the molecular identification of a glucose permease from Mycobacterium smegmatis,a model organism for our understanding of the life patterns of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae. A computer-based search of the available genome of M. smegmatis mc(2) 155 with the sequences of well-characterized glucose transporters revealed the gene msmeg4187 as a possible candidate. The deduced protein belongs to the major facilitator superfamily of proton symporters and facilitators and exhibits up to 53% of amino acid identity to other members of this family. Heterologous expression of msmeg4187 in an Escherichia coli glucose-negative mutant led to the restoration of growth on glucose. The determination of the biochemical features characterize MSMEG4187 (GlcP) as a high affinity (K(m) of 19 microM), glucose-specific permease. The results represent the first molecular characterization of a sugar permease in mycobacteria, and thus supply fundamental data for further in-depth analysis on the nutritional lifestyle of these bacteria.

Emergence of species-specific transporters during evolution of the hemiascomycete phylum.

We have traced the evolution patterns of 2480 transmembrane transporters from five complete genome sequences spanning the entire Hemiascomycete phylum: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica. The use of nonambiguous functional and phylogenetic criteria derived from the TCDB classification system has allowed the identification within the Hemiascomycete phylum of 97 small phylogenetic transporter subfamilies comprising a total of 355 transporters submitted to four distinct evolution patterns named "ubiquitous," "species specific," "phylum gains and losses," or "homoplasic." This analysis identifies the transporters that contribute to the emergence of species during the evolution of the Hemiascomycete phylum and may aid in establishing novel phylogenetic criteria for species classification.

Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residues (C-less permease), Asp237 and Lys358 were replaced with Cys or other amino acids to pursue the proposal that the two residues form a charge pair [King, S. C., Hansen, C. L., & Wilson, T.H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Individual replacement of Asp237 with Cys, Ala, or Lys or replacement of Lys358 with Cys, Ala, or Asp virtually abolishes active lactose transport. However, simultaneous replacement of both residues with Cys and/or Ala yields permease with high activity. Therefore, neutral amino acid substitutions at either position are detrimental only because they leave the opposing charge unpaired. Strikingly, moreover, when Asp237 is interchanged with Lys358, high activity is observed. The results indicate strongly that Asp237 and Lys358 interact to form a salt bridge and that neither residue nor the salt bridge per se is important for activity. Immunoblots reveal low membrane levels of the active mutants lacking the putative salt bridge, suggesting a role for the salt bridge in either permease folding or stability and raising the possibility that the salt bridge may exist in a folding intermediate but not in the mature protein. Remarkably, however, a mutant with Cys in place of Asp237 is restored to full activity by carboxymethylation which recreates a negative charge at position 237. Pulse-chase analysis and heat-inactivation studies indicate that the stability of the double mutant with Cys at positions 237 and 358 is comparable to C-less. Therefore, the interaction between Asp237 and Lys358 is likely to be important for permease folding and is maintained in the mature protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.