Biotransformations of aromatic aldehydes by acetogenic bacteria

Vanillin was subject to O demethylation and supported growth of Clostridium formicoaceticum and Clostridium thermoaceticum. Vanillin was also stimulatory to the CO-dependent growth of Peptostreptococcus productus. The aldehyde substituent of vanillin was metabolized by routes which were dependent upon both the acetogen and a co-metabolizable substrate (e.g. carbon monoxide [CO]). C. formicoaceticum and C. thermoaceticum oxidized the aldehyde group of vanillin to the carboxyl level, while P. productus reduced the aldehyde group of vanillin to the alcohol level. In contrast, during CO-dependent growth, C. thermoaceticum reduced 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol while P. productus both reduced and oxidized 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol and 4-hydroxybenzoate, respectively. These metabolic potentials indicate aromatic aldehydes may affect the flow of reductant during acetogenesis.     

Sulfite reduction in mycobacteria

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k(cat) = 1.0 (+/-0.1) electron consumed per second, and K(m(SO(3)(-2))) = 27 (+/-1) microM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe(2+) into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.     

Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria

Successful treatment of human tuberculosis requires 6–9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms – organized communities of surface-attached cells – but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.     

Generation of CD8+ T-cell responses by a recombinant nonpathogenic Mycobacterium smegmatis vaccine vector expressing human immunodeficiency virus type 1 Env

Because the vaccine vectors currently being evaluated in human populations all have significant limitations in their immunogenicity, novel vaccine strategies are needed for the elicitation of cell-mediated immunity. The nonpathogenic, rapidly growing mycobacterium Mycobacterium smegmatis was engineered as a vector expressing full-length human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope protein. Immunization of mice with recombinant M. smegmatis led to the expansion of major histocompatibility complex class I-restricted HIV-1 epitope-specific CD8(+) T cells that were cytolytic and secreted gamma interferon. Effector and memory T lymphocytes were elicited, and repeated immunization generated a stable central memory pool of virus-specific cells. Importantly, preexisting immunity to Mycobacterium bovis BCG had only a marginal effect on the immunogenicity of recombinant M. smegmatis. This mycobacterium may therefore be a useful vaccine vector.     

A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.     

Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells

The survival and persistence of Mycobacterium tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not been demonstrated or elucidated. Using a novel gain-of-function genetic screen, we demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Expression of M. tuberculosis nuoG in nonpathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected human or mouse macrophages, and increased their virulence in a SCID mouse model. Conversely, deletion of nuoG in M. tuberculosis ablated its ability to inhibit macrophage apoptosis and significantly reduced its virulence in mice. These results identify a key component of the genetic basis for an important virulence trait of M. tuberculosis and support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis.