African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali,Nigeria, Cameroon,and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.Mycobacterium bovis causes bovine tuberculosis (TB), an important disease of domesticated cattle that has a major economic and health impact throughout the world (61, 64, 65). The pathogen is a member of the Mycobacterium tuberculosis complex, which includes many species and subspecies that cause similar pathologies in a variety of mammalian hosts. The most notable member of the complex is M. tuberculosis, the most important bacterial pathogen of humans. In contrast to M. tuberculosis, which is largely host restricted to humans, M. bovis is primarily maintained in bovids, in particular, domesticated cattle, although the pathogen can frequently be recovered from other mammals, including humans (61). Bovine TB is found in cattle throughout the world and has been reported on every continent where cattle are farmed (3).Bovine TB has been reduced or eliminated from domestic cattle in many developed countries by the application of a test-and-cull policy that removes infected cattle (3, 8, 16, 17, 61, 64, 65). However, in Africa, although bovine TB is known to be common in both cattle and wildlife, control policies have not been enforced in many countries due to cost implications, lack of capacity, and infrastructure limitations (8, 16, 17, 57). In 1998, Cosivi et al. reported of bovine TB, “Of all nations in Africa, only seven apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease; the remaining 48 control the disease inadequately or not at all” (16). In the intervening years, the situation is not thought to have improved (8); however, preliminary surveys of bovine TB have been carried out in some African countries (4, 7, 12, 37, 44, 49, 53, 54, 56).The most common epidemiological molecular-typing method applied to strains of M. bovis is spoligotyping. This method identifies polymorphism in the presence of spacer units in the direct-repeat (DR) region in strains of the M. tuberculosis complex (36, 67). The DR is composed of multiple, virtually identical 36-bp regions interspersed with unique DNA spacer sequences of similar size (direct variant repeat [DVR] units). Spacer sequences are unique to the DR region, and copies are not located elsewhere in the chromosome (68). The DR region may contain over 60 DVR units; however, 43 of the spacer units were selected from the spacer sequences of the M. tuberculosis reference strain H37Rv and M. bovis BCG strain P3 and are used in the standard application of spoligotyping to strains of the M. tuberculosis complex (29, 36). The DR region is polymorphic because of the loss (deletion) of single or multiple spacers, and each spoligotype pattern from strains of M. bovis is given an identifier (http://www.Mbovis.org).Several studies of the DR regions in closely related strains of M. tuberculosis have concluded that the evolutionary trend for this region is primarily loss of single DVRs or multiple contiguous DVRs (22, 29, 68); duplication of DVR units or point mutations in spacer sequences were found to be rare. The loss of discrete units observed by Groenen et al. (29) led them to suggest that the mechanism for spacer loss was homologous recombination between repeat units. However, a study by Warren et al. (69) suggested that for strains of M. tuberculosis, insertion of IS6110 sequences into the DR region and recombination between adjacent IS6110 elements were more important mechanisms for the loss of spacer units.The population structure of the M. tuberculosis group of organisms is apparently highly clonal, without any transfer and recombination of chromosomal sequences between strains (15, 30, 60, 61). In a strictly clonal population, the loss by deletion of unique chromosomal DNA cannot be replaced by recombination from another strain, and the deleted region will act as a molecular marker for the strain and all its descendants. Deletions of specific chromosomal regions (regions of difference [RDs] or large sequence polymorphisms) have been very successful at identifying phylogenetic relationships in the M. tuberculosis complex (11, 25, 26, 35, 48, 50, 61, 62, 66). However, because the loss of spoligotype spacer sequences is so frequent, identical spoligotype patterns can occur independently in unrelated lineages (homoplasy), and therefore, the deletion of spoligotype spacers may be an unreliable indicator of phylogenetic relationship (61, 69).In samples of M. bovis strains from Cameroon, Nigeria, Chad, and Mali, spoligotyping was used to show that many of the strains had similar spoligotype patterns that lacked spacer 30, and it has been suggested that strains from these four countries are phylogenetically related (12, 18, 49, 53). We have extended the previous observations of spoligotype similarities between strains from these countries and confirmed the existence of a unique clonal complex of M. bovis, all descended from a single strain in which a specific deletion of chromosomal DNA occurred. We have named this clonal complex of M. bovis strains African 1 (Af1), and we show that this clonal complex is dominant in these four west-central African countries but rare in eastern and southern Africa. Extended genotyping, using variable-number tandem repeats (VNTR), of strains with the most common spoligotype patterns suggests that each of these four west-central African countries has a unique population structure. Evolutionary scenarios that may have led to the present day distribution of the Af1 clonal complex are discussed.     

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N²-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism''s response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19, 21, 60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family polymerases, whose founder member is Escherichia coli Pol IV (63), is conserved among all domains of life (44). The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N²-dG adducts (27, 28, 34, 40, 45, 67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).The M. tuberculosis genome encodes two Y family polymerase homologs belonging to the DinB subfamily, designated herein as DinB1 (DinX, encoded by Rv1537) and DinB2 (DinP, encoded by Rv3056), as well as a third, distantly related homolog encoded by Rv3394c (see Fig. S1 in the supplemental material) (9). On the basis of sequence similarity with their counterparts from E. coli (63) and Pseudomonas aeruginosa (54), including the complete conservation of key acidic residues essential for catalysis, DinB1 and DinB2 may be functional DNA polymerases (see Fig. S1). In contrast, Rv3394c lacks these residues and as such is unlikely to have polymerase activity (see Fig. S1). Unlike most Y family polymerase-encoding genes investigated with other bacteria (17, 26, 54, 58), dinB1 and dinB2 expression in M. tuberculosis is not dependent on RecA, the SOS response, or the presence of DNA damage (5, 7, 52). That these genes are regulated by other mechanisms and so may serve distinct roles in DNA metabolism in M. tuberculosis is suggested by the observation that dinB1 is differentially expressed in pulmonary TB (51) and is a member of the SigH regulon (30), whereas expression of dinB2 is induced following exposure to novobiocin (5).In this study, we adopted a genetic approach to investigate the function of dinB1 and dinB2 in M. tuberculosis. Mutants with altered complements or expression levels of dinB1 and/or dinB2 were analyzed in vitro and in vivo under conditions predicted to be phenotypically revealing based on DinB function established with other model organisms. The lack of discernible phenotypes in any of the assays employed suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.     

Mycobacterium tuberculosis Is Able To Accumulate and Utilize Cholesterol

It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.Mycobacterium tuberculosis, the causative agent of tuberculosis, is a very successful pathogen that infects one-third of the human population (21). Only 10% of primary infected individuals develop active disease during their lifetimes. Tubercle bacilli are able to persist in a dormant state, from which they may reactivate and induce the contagious disease state (13). In asymptomatic hosts, M. tuberculosis exists in reservoirs called granulomas, which are cellular aggregates that restrict bacterial spreading (40). Granulomas are organized collections of mature macrophages that exhibit a certain typical morphology and that arise in response to persistent intracellular pathogens (1, 4). Pathogenic mycobacteria can induce the formation of foamy macrophages filled with lipid-containing bodies; these have been postulated to act as a secure, nutrient-rich reservoir for tubercle bacilli (31). Moreover, M. tuberculosis DNA has been detected in fatty tissues surrounding the kidneys, as well as those of the stomach, lymph nodes, heart, and skin. Tubercle bacilli are able to enter adipocytes, where they accumulate within intracytoplasmic lipid inclusions and survive in a nonreplicating state (26). In vivo, it is expected that M. tuberculosis adapts metabolically to nutrient-poor conditions characterized by glucose deficiency and an abundance of fatty acids (25, 26). The presence of a complex repertoire of lipid metabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important alternative carbon and energy sources for this pathogen (7).One attractive potential alternative nutrient that is readily available in the mammalian host is cholesterol, a major sterol of the plasma membrane. The presence of cholesterol in lipid rafts is required in order for microorganisms to enter the intracellular compartment (14). Studies have shown that cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found to accumulate at the site of M. tuberculosis entry (2, 12, 30). Moreover, cholesterol depletion overcomes the phagosome maturation block experienced by Mycobacterium avium-infected macrophages (10).It is well known that cholesterol can be utilized by fast-growing, nonpathogenic mycobacteria (5, 20, 22), but it was previously thought that pathogenic mycobacteria might not be able to use cholesterol as a carbon and energy source (3). Recently, however, bioinformatic analysis identified a cassette of cholesterol catabolism genes in actinomycetes, including the M. tuberculosis complex (41). Microarray analysis of Rhodococcus sp. grown in the presence of cholesterol revealed the upregulation of 572 genes, most of which fell within six clearly discernible clusters (41). Most of the identified genes had significant homology to known steroid degradation genes from other organisms and were distributed within a single 51-gene cluster that appears to be very similar to a cluster present in the genome of M. tuberculosis (41). Many of the cholesterol-induced genes had been previously selected by transposon site hybridization analysis of genes that are essential for survival of tubercle bacilli (33) and/or are upregulated in gamma interferon-activated macrophages (37, 42). It was also demonstrated that the M. tuberculosis complex can grow on mineral medium with cholesterol as a primary source of carbon (27, 41). Moreover, the growth of tubercle bacilli on cholesterol was significantly affected by knockout of the mce4 gene, which encodes an ABC transporter responsible for cholesterol uptake (24, 27). Earlier studies had shown that disruption of mce4 attenuated bacterial growth in the spleens of infected animals that had developed adaptive immunity (17, 35).In the present study, we demonstrate for the first time that M. tuberculosis utilizes cholesterol via the 4-androstene-3,17-dione/1,4-androstadiene-3,17-dione pathway (AD/ADD) and that this process requires production of an intact KstD enzyme. We also show that tubercle bacilli growing in medium containing an alternative carbon source can accumulate cholesterol in the free-lipid zone of their cell walls, and this accumulation affects cell wall permeability.     

Influence of Environmental Gradients on the Abundance and Distribution of Mycobacterium spp. in a Coastal Lagoon Estuary

Environmental mycobacteria are of increasing concern in terms of the diseases they cause in both humans and animals. Although they are considered to be ubiquitous in aquatic environments, few studies have examined their ecology, and no ecological studies of coastal marine systems have been conducted. This study uses indirect gradient analysis to illustrate the strong relationships that exists between coastal water quality and the abundance of Mycobacterium spp. within a U.S. mid-Atlantic embayment. Mycobacterium species abundance and water quality conditions (based on 16 physical and chemical variables) were examined simultaneously in monthly samples obtained at 18 Maryland and Virginia coastal bay stations from August 2005 to November 2006 (n = 212). A quantitative molecular assay for Mycobacterium spp. was evaluated and applied, allowing for rapid, direct enumeration. By using indirect gradient analysis (environmental principal-components analysis), a strong linkage between eutrophic conditions, characterized by low dissolved-oxygen levels and elevated nutrient concentrations, and mycobacteria was determined. More specifically, a strong nutrient response was noted, with all nitrogen components and turbidity measurements correlating positively with abundance (r values of >0.30; P values of <0.001), while dissolved oxygen showed a strong negative relationship (r = −0.38; P = 0.01). Logistic regression models developed using salinity, dissolved oxygen, and total nitrogen showed a high degree of concordance (83%). These results suggest that coastal restoration and management strategies designed to reduce eutrophication may also reduce total mycobacteria in coastal waters.Environmental mycobacteria, or nontuberculous mycobacteria (NTM), include all species of mycobacteria other than those in the Mycobacterium tuberculosis complex and M. leprae. In general, NTM are aerobic, acid-fast, gram-positive, non-spore-forming, nonmotile organisms found as free-living saprophytes in soil and water (12, 14, 20, 21, 35). However, several members of this group can cause serious disease in humans, including pulmonary infections, cervical lymphadenitis, ulcerative necrosis, skin infections, and disseminated infections associated primarily with autoimmune disorders (12, 29). For example, disseminated infection with the Mycobacterium avium complex can occur in up to 40% of late-stage AIDS patients in developed countries (43). NTM can also have costly and problematic effects on wild and domesticated animals (17, 23). Thus, understanding the sources and reservoirs of these bacteria has become a priority in recent years (12, 34).While the mode of infection has been poorly established for many cases involving NTM, water is commonly implicated as either a source or a vector (12, 43). NTM are considered to be ubiquitous in the environment and have been cultured globally from samples obtained from freshwaters and marine natural waters (12), swimming pools and hot tubs (11, 25), and drinking water supplies (12, 13), among others. However, only a limited number of attempts have been made to examine the association of their distribution and abundance with environmental parameters (1, 21, 24). The abundance of the M. avium complex was found to correlate positively with water temperature and levels of zinc and humic and fulvic acids and negatively with the dissolved-oxygen content and pH in brown-water swamps in the southeastern United States (24). In a study of Finnish brook waters, acidic conditions, along with the presence of peatlands, chemical oxygen demand, increased precipitation, water color, and concentrations of several metals, were found to favor total NTM (20, 21). However, recent efforts with samples from the Rio Grande River in the United States found positive correlations with the presence of coliforms and Escherichia coli counts and negative correlations with chemical toxicity and water temperature in this alkaline, oligotrophic system (1). Although system-specific differences may be apparent, no attempts to examine mycobacterial ecology in marine and estuarine systems have been reported to date.Historically, researchers have relied on culture-based techniques for detection and enumeration of mycobacteria from environmental samples (1, 20, 21, 43). Because of the slow growth of many mycobacteria, culture from environmental samples requires decontamination, which can severely impact both the quantity and diversity of species recovered (18, 19). Recently, quantitative PCR (qPCR) has gained favor as a means of rapidly enumerating organisms or genes in environmental samples (5, 15, 38, 40). This method allows for the continuous monitoring of the reaction through the use of fluorescent reporter molecules or DNA stains. Because of this strategy, the reaction can be evaluated at the peak of the exponential phase, reducing errors of reagent depletion and assay efficiency associated with end point reads. Quantification is based on the principle that the amount of the starting template is directly proportional to the number of cycles required to reach the peak of the exponential phase, and is evaluated through the preparation of standards.Like many coastal lagoon estuaries, the shallow embayments bordering the Maryland and Virginia seaboard are highly susceptible to anthropogenic influence, as they are visited by millions of people annually for vacation and water-related recreation (44). While eutrophication and degraded environmental conditions have been generally linked to factors or organisms which can ultimately influence human health, little attention has been given to the response of bacteria (16, 45). In this paper, we describe our efforts to examine environmental influences on the abundance and distribution of NTM in a dynamic estuarine system.     

Mycobacterial Biofilms Facilitate Horizontal DNA Transfer between Strains of Mycobacterium smegmatis

Conjugal transfer of chromosomal DNA between strains of Mycobacterium smegmatis occurs by a novel mechanism. In a transposon mutagenesis screen, three transfer-defective insertions were mapped to the lsr2 gene of the donor strain mc²155. Because lsr2 encodes a nonspecific DNA-binding protein, mutations of lsr2 give rise to a variety of phenotypes, including an inability to form biofilms. In this study, we show that efficient DNA transfer between strains of M. smegmatis occurs in a mixed biofilm and that the process requires expression of lsr2 in the donor but not in the recipient strain. Testing cells from different strata of standing cultures showed that transfer occurred predominantly at the biofilm air-liquid interface, as other strata containing higher cell densities produced very few transconjugants. These data suggest that the biofilm plays a role beyond mere facilitation of cell-cell contact. Surprisingly, we found that under standard assay conditions the recipient strain does not form a biofilm. Taking these results together, we conclude that for transfer to occur, the recipient strain is actively recruited into the biofilm. In support of this idea, we show that donor and recipient cells are present in almost equal numbers in biofilms that produce transconjugants. Our demonstration of genetic exchange between mycobacteria in a mixed biofilm suggests that conjugation occurs in the environment. Since biofilms are considered to be the predominant natural microhabitat for bacteria, our finding emphasizes the importance of studying biological and physical processes that occur between cells in mixed biofilms.Biofilms are dynamic communities of microorganisms that form on surfaces or at air-liquid interfaces (17, 20, 41). They arise following the attachment of bacteria to a surface; the bacteria then grow, differentiate, and multiply. The colonizing bacteria produce extracellular polymers, which encapsulate the cells and trap particulate matter, nutrients, and other bacteria that in turn contribute to the further development of the biofilm. Thus, as the biofilm develops it becomes increasingly heterogeneous. Microbial life is thought to exist predominantly in a biofilm, and biofilms can have either beneficial or harmful impacts on their environments (23). From a medical standpoint, biofilms can create serious problems. Bacteria within a biofilm are inherently more resistant to antibiotics, which makes their eradication difficult and is particularly problematic for patients with surgical implants resulting in chronic infections (19, 33).Mycobacteria are known to form biofilms; however, relatively little is known about the mechanism of biofilm formation and development or its role in the biology of Mycobacterium species. For practical reasons, most biofilm studies have focused on the more rapidly growing and less pathogenic species, namely, Mycobacterium fortuitum, M. marinum, and M. smegmatis (16, 18, 36). In particular, genetic studies of M. smegmatis have provided insight into some of the key factors required for biofilm formation (5, 30, 31, 36, 37). Glycopeptidolipids are required for initial surface attachment of M. smegmatis, while GroEL1 is required for a later stage of biofilm development. GroEL1 is thought to coordinate a switch in mycolic acid synthesis from very-long-chain (C₇₀ to C₉₀) to shorter-chain (C₅₆ to C₆₈) derivatives. The short-chain mycolic acids were proposed previously to form the extracellular matrix critical for biofilm formation (30). The metabolic switch in mycolic acid synthesis was also correlated with iron availability. Under iron-limiting conditions or in exochelin mutants, biofilm formation is arrested, an event coincident with the synthesis of short-chain mycolic acids (31).A cytoplasmic protein, Lsr2, has also been shown to be critical in biofilm formation (5, 8). Lsr2 was first described as an immunodominant antigen of M. leprae (24); however, it has since been shown to modulate a diverse range of processes. The resultant phenotypes of lsr2 mutants can be attributed to the ability of Lsr2 to bind DNA nonspecifically (6, 7, 15). Lsr2 belongs to the family of histone-like DNA binding proteins, a fact that was demonstrated by showing that lsr2 can suppress hns mutant phenotypes in Escherichia coli and that hns can suppress lsr2 mutant phenotypes in M. smegmatis (14). lsr2 mutants have an altered colony morphology and are defective in biofilm formation (2, 5, 8). This phenotype is presumably a consequence of the altered expression of key surface proteins and apolar lipids, such as mycolyl-diacylglycerols, which are lacking in lsr2 mutants (5). In this study, we show that mycobacterial conjugal DNA transfer requires Lsr2 and that genetic exchange occurs in a mixed biofilm.We have previously described a novel conjugation system in M. smegmatis (34). Chromosomal transfer occurs in a unidirectional fashion from a donor to a recipient, and this process requires prolonged cell-cell contact (47). Our transfer studies to date have established that the genetic requirements differ markedly between the donor and recipient strains. Because bioinformatic searches of the completed M. smegmatis donor genome have failed to identify obvious transfer-related genes, transposon mutagenesis screens were used to empirically identify donor and recipient genes involved in DNA transfer. A transposon mutagenesis screen of the recipient strain identified loci throughout the genome that were necessary for efficient transfer (9). In contrast, mutagenesis screens of the donor strain failed to identify transfer-defective mutants; instead, hyperconjugative donor mutants were found (12). The hyperconjugative mutations mapped to the esx-1 locus, which encodes a highly conserved secretory apparatus (ESX-1) that is required for full virulence of M. tuberculosis (1, 10), as well as for DNA transfer in the recipient M. smegmatis (9). The hyperconjugative phenotype of esx-1 donor mutants indicated that protein secretion negatively regulates conjugal transfer from the donor.We have exploited the hyperconjugative phenotype of esx-1 mutants so as to increase the sensitivity of a genetic screen for transfer-defective mutants (29). This strategy resulted in the identification of lsr2 as being important for DNA transfer in the donor and led us to investigate the dependence of conjugation on biofilm formation. We show here that stationary liquid cultures develop a surface biofilm in which DNA transfer rates approach those found in our established solid-medium mating assays. Our data further suggest that the biofilm contributes in more ways than merely providing a concentrated cell environment, given that dense cell aggregates resting on the bottoms of these same stationary cultures are transfer deficient. The prevalence of heterogeneous, mixed biofilms in natural environments suggests that mycobacterial conjugal DNA transfer may occur outside the laboratory.     

Characterization of Mycobacterium leprae RecA Intein, a LAGLIDADG Homing Endonuclease, Reveals a Unique Mode of DNA Binding, Helical Distortion, and Cleavage Compared with a Canonical LAGLIDADG Homing Endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of the M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg²⁺ or Mn²⁺. A combination of approaches, including four complementary footprinting assays such as DNase I, copper-phenanthroline, methylation protection, and KMnO₄, enhancement of 2-aminopurine fluorescence, and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site, and generates two staggered double strand breaks. Taken together, these results implicate that PI-MleI possesses a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of the LAGLIDADG family of homing endonucleases.Mycobacterium leprae, a Gram-positive rod-shaped bacillus, mostly found in warm tropical countries, is the bacterium that causes leprosy in humans (1). The lack of understanding of the basic biology of M. leprae is believed to be the key factor for the failure of leprosy research to advance. The genome sequence of M. leprae contains 3.27 Mb and has an average G + C content of 57.8%, values much lower than the corresponding values for Mycobacterium tuberculosis, which are ∼4.41 Mb and 65.6% G + C, respectively (2). There are some 1500 genes that are common to both M. leprae and M. tuberculosis. The comparative genome analysis suggests that both species of mycobacteria are derived from a common ancestor and, at one stage, had gene pools of similar size. The downsizing of the M. tuberculosis genome from ∼4.41 to 3.27 Mb of M. leprae would account for the loss of some 1200 protein-coding sequences (1, 3). There is evidence that many of the genes that were present in the genome of M. leprae have truly been lost (1, 3). Comparative genomics of M. leprae with that of M. tuberculosis indicate that the former has undergone substantial downsizing, losing more than 2000 genes, thus suggesting an extreme case of reductive evolution in a microbial pathogen (1). With the availability of the M. leprae genome sequence, using functional genomics approaches, it is possible to identify the gene products, elucidate the mechanism of their action, and identify novel drug targets for rational design of new therapeutic regimens and drugs to treat leprosy.Eubacterial RecA proteins catalyze a set of biochemical reactions that are essential for homologous recombination, DNA repair, restoration of stalled replication forks, and SOS response (4–7). RecA protein and the process of homologous recombination, which is the main mechanism of genetic exchange, are evolutionarily conserved among a range of organisms (4, 7). Perhaps the most striking development in the field of RecA protein biology was the discovery of an in-frame insertion of an intein-coding sequence in the recA genes of M. tuberculosis and M. leprae (8, 9). In these organisms, RecA is synthesized as a large precursor, which undergoes protein splicing to excise the intein, and the two flanking domains called exteins are ligated together to generate a functionally active RecA protein (9, 10). The milieu in which RecA precursor undergoes splicing differs substantially between M. tuberculosis and M. leprae. M. leprae RecA precursor (79 kDa) undergoes splicing only in mycobacterial species, whereas M. tuberculosis RecA precursor (85 kDa) is spliced efficiently in Escherichia coli as well (9–11). Intriguingly, M. tuberculosis and M. leprae RecA inteins differ greatly in their size, primary sequence, and location within the recA gene, thereby suggesting two independent origins during evolution (9). The occurrence of inteins in the obligate mycobacterial pathogens, M. tuberculosis, M. leprae, and Mycobacterium microti, suggested that RecA inteins might play a role in mycobacterial functions related to pathogenesis or virulence (9). Previously, we have shown that M. tuberculosis RecA intein (PI-MtuI),² which contains Walker A motif, displays dual target specificity in the presence of alternative cofactors in an ATP-dependent manner (12, 13).Since their discovery in Saccharomyces cerevisiae (14, 15), a large number of putative homing endonucleases have been found in a diverse range of proteins in all the three domains of life (16–19). The majority of inteins possess the protein splicing and homing endonuclease activities (18, 19). Homing endonucleases are a class of diverse rare-cutting enzymes that promote site-specific transposition of their encoding genetic elements by inflicting double-stranded DNA breaks via different cleavage mechanisms in alleles lacking these elements (18–23). In addition, these are characterized by their ability to bind long DNA target sites (14–40 bp), and their tolerance of minor sequence changes in their binding region. These have been divided into highly divergent subfamilies on the basis of conserved sequence and structural motifs as follows: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and the more recently identified PD(D/E)XK families (18–24). LAGLIDADG homing enzymes, which include the largest family, contain one or two copies of the conserved dodecapeptide motif and utilize an extended protein-DNA interface covering up to 40 bp to acquire their necessary specificity (18–22). The LAGLIDADG sequence is a part of the conserved 10- or 12-residue sequence motif defining the family of LAGLIDADG-type homing endonucleases; therefore, it is designated as deca- or dodecapeptide motif (19).Comparison of the M. leprae recA intervening sequence with known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease (25, 26). In light of massive gene decay and function loss in the leprosy bacillus, and dissimilarities in size and primary structures among mycobacterial inteins, we sought to investigate whether M. leprae recA intervening sequence encodes a catalytically active homing endonuclease. In this study, we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations Mg²⁺ or Mn²⁺. Furthermore, using a variety of approaches, we have mapped the positions of PI-MleI binding as well as cleavage in the cognate DNA, thus providing the most comprehensive analysis of PI-MleI. Taken together, these results suggest that PI-MleI possesses a modular structure with functionally separable domains for DNA target recognition and cleavage, each with distinct sequence preferences. These results provide insights into understanding the function and evolution of the family of LAGLIDADG homing endonucleases.     

Interaction of Mycobacterium ulcerans with Mosquito Species: Implications for Transmission and Trophic Relationships

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease that causes significant morbidity in Africa and Australia. Person-to-person transmission of Buruli ulcer is rare. Throughout Africa and Australia infection is associated with residence near slow-moving or stagnant water bodies. Although M. ulcerans DNA has been detected in over 30 taxa of invertebrates, fish, water filtrate, and plant materials and one environmental isolate cultured from a water strider (Gerridae), the invertebrate taxa identified are not adapted to feed on humans, and the mode of transmission for Buruli ulcer remains an enigma. Recent epidemiological reports from Australia describing the presence of M. ulcerans DNA in adult mosquitoes have led to the hypothesis that mosquitoes play an important role in the transmission of M. ulcerans. In this study we have investigated the potential of mosquitoes to serve as biological or mechanical vectors or as environmental reservoirs for M. ulcerans. Here we show that Aedes aegypti, A. albopictus, Ochlerotatus triseriatus, and Culex restuans larvae readily ingest wild-type M. ulcerans, isogenic toxin-negative mutants, and Mycobacterium marinum isolates and remain infected throughout larval development. However, the infections are not carried over into the pupae or adult mosquitoes, suggesting an unlikely role for mosquitoes as biological vectors. By following M. ulcerans through a food chain consisting of primary (mosquito larvae), secondary (predatory mosquito larva from Toxorhynchites rutilus septentrionalis), and tertiary (Belostoma species) consumers, we have shown that M. ulcerans can be productively maintained in an aquatic food web.Infection with Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is associated with residence near stagnant and slow-moving water bodies in areas in which the disease is endemic (5, 36, 40, 45, 50). A plasmid-encoded macrolide toxin, mycolactone, is the primary virulence determinant of M. ulcerans (8, 41). Biting aquatic insects, such as several taxa in the Belostomatidae and Naucoridae families (Hemiptera), have been suggested as possible vectors of M. ulcerans in several laboratory experiments (16, 19, 20, 24, 31, 32); however, there is little empirical evidence from field studies to support the contention that these biting insects vector M. ulcerans to humans (2). In Melbourne, Australia, recent epidemiological evidence suggests that mosquitoes may serve as vectors in the transmission of BU disease (10, 11, 12, 34, 35). In this study, 957 pools consisting of over 11,000 mosquitoes of four different species were collected and tested by quantitative PCR (qPCR) for the presence of M. ulcerans DNA, and positive results were obtained from 48 of 957 pools tested (10). Of the 48 positive pools, 13 were positive for PCR directed against two insertion sequences (IS2404 and IS2606) as well as against sequence based on the ketoreductase domain of the mycolactone toxin genes. Because all of these target sequences are present multiple times in the genome, it was difficult to assign genome equivalents to these results. However, data from laboratory experiments suggested that 10 to 100 M. ulcerans isolates per mosquito were present in the positive pools. Epidemiological work also suggested a seasonal relationship between Buruli ulcer and mosquito-vectored diseases in Australia (12). These studies are extremely provocative and raise a number of questions for further work. What is the prevalence of M. ulcerans in other invertebrate taxa in the same environment? What is the infection rate in equal numbers of mosquitoes collected from areas in which the disease is not endemic? Is it possible to obtain physical evidence for the presence of M. ulcerans through microscopy or culture of mosquitoes in areas in which the disease is endemic, and, finally, what can we learn from laboratory studies concerning the interaction between mosquitoes and M. ulcerans?The recent work from Australia suggesting that M. ulcerans is spread by mosquitoes is particularly significant because adult mosquitoes are the most important group of insects in the spread of human disease. They may serve as biological vectors that provide a major environment for pathogen replication, as in malaria or yellow fever, or as mechanical vectors that carry organisms between hosts without serving as a site of replication (1, 4, 7, 9, 38). Larval mosquitoes are common in habitats associated with BU disease, most notably lentic or standing water habitats, and feed by filtering particles in the water using labral head fans (21). Members of some genera (i.e., Anopheles) aggregate at the air-water interface in microlayers near plant stems and algal mats (27, 28, 46), where they feed on microorganisms such as bacteria and algae (47). Because of their collecting-filtering feeding mode, there is potential for larvae to consume M. ulcerans and concentrate mycobacteria through their feeding activities (22, 23).In Ghana, the occurrence of M. ulcerans among invertebrate communities in lentic habitats has been documented from regions in Ga West and Ga East Districts in which the disease is endemic as well as those in which it is not endemic (2, 49) but not in geographically distinct areas in which the disease is not endemic such as the Volta region (49). M. ulcerans has been identified in a suite of environmental samples such as filtered water, biofilms, and algae as well as among a broad spectrum of invertebrate taxa, including both larval and adult mosquitoes (2, 11, 17, 49). However, the replication and trophic movement of M. ulcerans within these environmental samples and invertebrate communities have not been experimentally investigated. Conceptual models have been proposed that assume that the primary consumers of M. ulcerans (e.g., mosquito larvae, cladocerans, and chironomid larvae) may feed on bacteria and algae in biofilms, filter suspended matter from the water column, and then initiate the passage of M. ulcerans through an aquatic food web (2, 22, 31). This model predicts the movement of M. ulcerans through secondary and tertiary consumers and implies a complex trophic relationship in the ecology of M. ulcerans as well as an important role of aquatic invertebrates in the disease ecology of M. ulcerans.In the studies reported here, we have explored the role of mosquitoes as biological or mechanical vectors of M. ulcerans, as well as the potential of mosquito larvae to play a central role in the movement of M. ulcerans through an aquatic food web. In order to investigate the ability of mosquito larvae to ingest and maintain M. ulcerans within their digestive tract as well as to persist throughout the mosquito development cycle, we took advantage of the fact that mosquito larvae naturally feed upon bacteria. Results presented here show that strains of M. ulcerans from Africa and Australia, as well as Mycobacterium marinum, were maintained at high levels in the larval mosquito gut for 6 days. However, neither M. ulcerans nor M. marinum was detected in adult mosquitoes that were infected in the larval stage. These results suggest that mosquitoes are unlikely to serve as biological vectors of M. ulcerans.We further developed a model for following the passage of M. ulcerans through a series of consumers to determine whether M. ulcerans could be passed up a trophic chain from primary to tertiary consumers. In this model, we conducted similar experiments using four species of nonpredatory mosquito larvae, Aedes aegypti (Linnaeus), Aedes albopictus (Skuse), Ochlerotatus triseriatus (Theobald), and Culex restuans (Theobald), as primary consumers. These larvae were infected with isogenic wild-type (WT) and toxin-negative isolates of M. ulcerans and of M. marinum, the closest relative to M. ulcerans (13, 14, 51). We have shown that M. ulcerans in mosquito larvae survive passage through secondary and tertiary consumers, thus providing the first laboratory evidence that M. ulcerans has the potential to move between and be maintained within different species in an aquatic food web.     

The Mycobacterium tuberculosis DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.Mycobacterium tuberculosis, a major human pathogen, infects nearly one-third of the people in the world and causes two million deaths per year (8). Most infections are latent, and a substantial number of new infections are transmitted by individuals in whom latent infections are being reactivated. Latency is a clinical term describing people that are infected with M. tuberculosis but lack symptoms of active disease. Traditionally, it has been thought that bacilli in latently infected individuals reside almost exclusively inside granulomas and mature tubercle lesions. Recent studies indicate that in latently infected individuals M. tuberculosis may also be found outside granulomas in places such as endothelial cells, fibroblasts, and adipose tissue (17, 28). The evidence for M. tuberculosis metabolic activity in vivo is more limited, but two studies by Lillebaek et al. are informative (24, 25). In these studies the researchers used detailed records of tuberculosis epidemiology and strain types in the fairly static population of Denmark. They found that strains isolated from patients thought to have reactivated disease (rather than a primary infection) were nearly identical to strains present 30 years earlier in the same geographic population. The near-identity of the strains and the fact that infections were attributed to reactivation suggest that bacteria in latently infected individuals experience little genetic change during years of latent infection. The researchers concluded that during latency, M. tuberculosis divides infrequently and is likely in a minimal metabolic state.One approach to study the M. tuberculosis metabolic state during latent infection is to use in vitro models that mimic conditions thought to exist in vivo. Such conditions include hypoxia produced in avascular calcified granulomas (40) and nitric oxide (NO) (27) or carbon monoxide (CO) (33) produced by activated immune cells. A widely used model is the “Wayne model” pioneered by Lawrence Wayne. In this model, a low-inoculum culture is sealed in a tube with stirring and allowed to slowly consume oxygen until the culture is anaerobic, resulting in a nonreplicating and apparently dormant state (45, 46). Another model used to look at dormant M. tuberculosis is a constant-hypoxia model that maintains a 0.2% oxygen tension in culture flasks (31).The common theme in these in vitro models used to obtain M. tuberculosis dormancy is inhibition of respiration. The DosR regulon is a set of at least 48 coregulated genes that are induced by three conditions that inhibit aerobic respiration: hypoxia, NO, and CO (42). Induction of the DosR regulon closely mirrors inhibition of respiration, indicating that control of the regulon is linked to the aerobic respiratory state of the bacilli (43). Several studies have shown that the DosR regulon is controlled by a three-component regulatory system composed of two sensor histidine kinases, DosS and DosT, and a response regulator, DosR (42). DosS and DosT both bind the respiration-impairing gases NO and CO (19, 20, 38), further supporting the hypothesis that the DosR regulon responds to, and is important during, conditions that do not allow aerobic respiration. Although the majority of the DosR-regulated genes have not been characterized, the timing of their induction combined with the conditions under which they respond suggests that they may play a role in adaptation of M. tuberculosis to its host environment. Consistent with this notion, DosR regulon genes are induced in the lungs of M. tuberculosis-infected mice (43), as well as in interferon-gamma-activated murine macrophages (34) and guinea pigs (37).Several studies have suggested that the DosR regulon plays a role in latent infection and in persistence in animal models that resemble human infection in some respects. Leyten et al. found that latently infected humans are more likely than humans with active infections to bear T cells specific for DosR regulon antigens (23), suggesting that the regulon is expressed during latency. Two recent studies confirmed that there is an immune response to DosR regulon antigens during latent infection (4, 36). Further evidence for clinical relevance in humans comes from a study showing that M. tuberculosis in sputum expresses the DosR regulon (15). The importance of this regulon for persistence in rabbit and guinea pig models was demonstrated by data showing a 2-log decrease in recovery of a DosR mutant 2 weeks (guinea pig) and 8 weeks (rabbit) after aerosol infection (11). A DosR mutant was also found to be significantly attenuated in guinea pig infection (26), further supporting the notion that the DosR regulon is required for persistence in vivo. It should be noted that in both studies showing the DosR phenotype (11, 26), full complementation and reversion to full virulence were not observed. However, it is now known that regulation of dosR expression is quite complex. Multiple regulatory sequences exist in and upstream of Rv3134c, the gene directly upstream of dosR (8). Failure to include such a regulatory sequence in a complemented strain would likely result in misregulation of dosR and poor complementation. Studies of DosR regulon mutants for murine infection have produced inconsistent findings that vary from hypervirulent (30) to attenuated (11) and not attenuated (3, 31). When animal models are compared, it is important to remember that M. tuberculosis-induced granulomas in primates, rabbits, and guinea pigs develop caseous necrosis and are hypoxic and/or anaerobic, while M. tuberculosis induced-granulomas in mice are neither hypoxic nor anaerobic (2, 21, 41). Furthermore, M. tuberculosis divides regularly in chronic murine infections (16), in contrast to the replication during latent infections, as demonstrated in the studies of Lillebaek et al. (24, 25). Such studies underscore the significant differences between models.A previous study with a DosR mutant in a closely related Mycobacterium bovis BCG strain showed that DosR expression is required for survival in an in vitro Wayne-like model of dormancy (5). Unexpectedly, two similar studies in M. tuberculosis did not show a strong survival defect for a DosR mutant (31, 43). The most recent study showed that there was only a modest survival defect in an H37Rv DosR mutant and concluded that the DosR regulon is a short-term phenomenon and is not responsible for the adaptation necessary to survive under primarily hypoxic conditions in vitro (31, 32).In this study we showed that the DosR regulon is required for M. tuberculosis survival during anaerobic dormancy. We also used a combination of genetic and biochemical approaches to demonstrate that this regulon is necessary to shift away from oxygen consumption, maintain ATP levels, and balance the redox state (NAD/NADH ratio) of the cell as oxygen becomes scarce. Furthermore, we showed that the DosR regulon is necessary for optimal transition of M. tuberculosis back to aerobic growth from an anaerobic or nitric oxide-induced nonrespiring state.     

Association of Missense Mutations in Epoxyalkane Coenzyme M Transferase with Adaptation of Mycobacterium sp. Strain JS623 to Growth on Vinyl Chloride

Vinyl chloride (VC) is a toxic groundwater pollutant associated with plastic manufacture and chlorinated solvent use. Aerobic bacteria that grow on VC as a carbon and energy source can evolve in the laboratory from bacteria that grow on ethene, but the genetic changes involved are unknown. We investigated VC adaptation in two variants (JS623-E and JS623-T) of the ethene-oxidizing Mycobacterium strain JS623. Missense mutations in the EtnE gene developed at two positions (W243 and R257) in cultures exposed to VC but not in cultures maintained on ethene. Epoxyalkane-coenzyme M transferase (EaCoMT) activities in cell extracts of JS623-E and JS623-T (150 and 645 nmol/min/mg protein, respectively) were higher than that of wild-type JS623 (74 nmol/min/mg protein), and in both variant cultures epoxyethane no longer accumulated during growth on ethene. The heterologous expression of two variant etnE alleles (W243G [etnE1] and R257L [etnE2]) from strain JS623 in Mycobacterium smegmatis showed that they had 42 to 59% higher activities than the wild type. Recombinant JS623 cultures containing mutant EtnE genes cloned in the vector pMV261 adapted to growth on VC more rapidly than the wild-type JS623 strain, with incubation times of 60 days (wild type), 1 day (pMVetnE1), and 35 days (pMVetnE2). The JS623(pMVetnE) culture did not adapt to VC after more than 60 days of incubation. Adaptation to VC in strain JS623 is consistently associated with two particular missense mutations in the etnE gene that lead to higher EaCoMT activity. This is the first report to pinpoint a genetic change associated with the transition from cometabolic to growth-linked VC oxidation in bacteria.Bacteria that biodegrade pollutants are useful for the cleanup of contaminated sites (i.e., bioremediation) and are interesting as models of evolutionary processes (21, 38, 40). Understanding the molecular genetic and evolutionary basis of biodegradation processes allows improved monitoring and predictions of bacterial activities in situ (39) and promises the development of improved strains and enzymes with increased specific activity (3), increased substrate affinity (16), extended substrate range (3, 16, 21, 37), extended inducer range (30, 31), or constitutive expression (39). Missense mutations in catabolic enzymes or regulatory proteins commonly lead to these changes (43), although other important mechanisms include duplication, deletion, and inversion (38-40).Vinyl chloride (VC) is a common groundwater pollutant (35) and known human carcinogen (24), and it poses a health risk to exposed populations. Although trace amounts (e.g., parts per trillion) of VC have been detected in uncontaminated soil (23), higher concentrations are found only associated with human industry, particularly the manufacture of polyvinylchloride (PVC) plastic and the chlorinated solvents trichloroethene (TCE) and perchloroethene (PCE) (4). Aerobic bacteria that grow on VC as a sole carbon and energy source are diverse, including strains of Mycobacterium (8, 17, 18), Nocardioides (8), Pseudomonas (11, 41, 42), Ochrobactrum (11), and Ralstonia (13, 33). The relative ease of the isolation of VC assimilators from chlorinated ethene-contaminated sites suggests that such bacteria are influential in the natural attenuation of VC, but this interpretation is complicated by the fact that VC-assimilating bacteria are closely related to ethene-assimilating bacteria (8-10, 29) and cannot yet be distinguished from them by molecular tests.The VC and ethene pathway and genes are homologous to some extent with the propene assimilation pathway and genes in Xanthobacter Py2 and Gordonia B-276. The comparison of the genomes of the VC-assimilating Nocardioides JS614 and the propene-assimilating Xanthobacter Py2 indicates that growth on alkenes requires about 20 kb of alkene/epoxide catabolic genes and approximately 7 kb of coenzyme M (CoM) biosynthesis genes. The oxidation of VC and ethene is initiated by an alkene monooxygenase (AkMO; EtnABCD) (8-10, 29), which yields epoxyethane from ethene and chlorooxirane from VC (8, 17). An epoxyalkane-coenzyme M transferase (EaCoMT) enzyme, EtnE, acts upon these reactive, toxic, and mutagenic epoxides (2, 19), converting them to hydroxyalkyl-CoM derivatives. The remainder of the VC/ethene pathway is unclear. The JS614 genome indicates further homology with propene oxidizers, in that a reductase/carboxylase and SDR family dehydrogenase are present, but that other aspects of the VC/ethene pathway gene cluster are unique (e.g., the presence of a semialdehyde dehydrogenase [5] and a disulfide reductase-like gene [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_008697","term_id":"119714007","term_text":"NC_008697"}}NC_008697]).The EtnE enzyme and the homologous XecA enzyme that acts on epoxypropane in Xanthobacter Py2 and Gordonia B-276 (9, 10, 12, 29) are unusual in their requirement for CoM as a cofactor. The C2- and C3-alkene oxidizers are the only Eubacteria known to biosynthesize and require CoM, which is otherwise found only in methanogenic Archaea. The XecA protein of Py2 has been purified and shown to be a Zn-dependent enzyme (1, 14, 26, 44). Based on sequence homology and the presence of the Cys-X-His-X_n-Cys motif (see Fig. S1 in the supplemental material), the EtnE enzymes also are likely to be Zn-dependent enzymes. Heterologous expression systems for XecA and EtnE have been developed (9, 25), but no crystal structures are available yet for EaCoMT from any source.Pure cultures of ethene-assimilating bacteria are capable of spontaneously adapting to growth on VC as a carbon source (22, 42), but the molecular basis of this phenomenon is not clear. This knowledge gap confounds the development of molecular probes specific for VC-assimilating bacteria. Pseudomonas aeruginosa strain DL1 shifted from cometabolism to growth on VC after more than 40 days of incubation (42), while Mycobacterium strains JS622, JS623, JS624, and JS625 took between 55 and 476 days to adapt to VC (22). The VC-adapted phenotype in Mycobacterium strains was not lost after growth in nonselective medium, suggesting a genetic change rather than a physiological adaptation (22).Here, we tested the hypothesis that mutations in the alkene/epoxide catabolic genes are responsible for VC adaptation. This was done by sequencing EtnEABCD genes in fosmid clones from cultures before and after VC adaptation, by sequencing etnE PCR products at different time points during VC adaptation, and by examining the EtnE enzyme activity in VC-adapted strains and recombinant strains carrying evolved etnE alleles.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.     

Novel Rhamnosyltransferase Involved in Biosynthesis of Serovar 4-Specific Glycopeptidolipid from Mycobacterium avium Complex

Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.The genus Mycobacterium has a unique feature in the cell envelope that contains a multilayered structure consisting of peptidoglycan, mycolyl-arabinogalactan complex, and surface glycolipids (8, 12). It is known that these components play a role in protection from environmental stresses, such as antimicrobial agents and host immune responses (8, 12). Some of them are recognized as pathogenic factors related to mycobacterial diseases, such as tuberculosis and leprosy (8, 12). In case of nontuberculous mycobacteria that are widely distributed in the natural environment as opportunistic pathogens, glycopeptidolipids (GPLs) are abundantly present on the cell envelope as surface glycolipids (34). GPLs have a core structure in which a fatty acyl-tetrapeptide is glycosylated with 6-deoxy-talose (6-d-Tal) and O-methyl-rhamnose (O-Me-Rha) (2, 5, 13). This structure is common to all types of GPLs, and GPLs with this structure that have not undergone further glycosylation are termed non-serovar-specific GPLs (nsGPLs) (2, 5, 13). Structural diversity generated by further glycosylations, such as rhamnosylation, fucosylation, and glucosylation, is observed for the oligosaccharide portion linked to the 6-d-Tal residue of nsGPLs from Mycobacterium avium complex (MAC), a member of the nontuberculous mycobacteria consisting of two species, M. avium and M. intracellulare (2, 5, 34). Consequently, these nsGPLs with varied oligosaccharides lead to the formation of the serovar-specific GPLs (ssGPLs) that define around 30 types of MAC serovars (10).The properties of MAC serovars are known to be notably different from each other and also to be closely associated with the pathogenicity of MAC (3, 6, 18, 30, 31, 32). Various epidemiological studies indicate that serovar 4 is the most prevalent type and is also one of the serovars frequently isolated from AIDS patients (1, 20, 33, 36). Additionally, pulmonary MAC disease caused by serovar 4 is shown to exhibit a poorer prognosis than that caused by other serovars (23). With respect to host immune responses to MAC infection, serovar 4-specific GPL is reported to have characteristic features that are in contrast to those of other ssGPLs (21, 30). Structurally, serovar 4-specific GPL contains a unique oligosaccharide in which the oligosaccharide of serovar 2-specific GPL is further glycosylated with 4-O-methyl-rhamnose (4-O-Me-Rha) residue through a 1→4 linkage (Table ​(Table1)1) (24). Therefore, it is thought that the presence of 4-O-Me-Rha and its linkage position are important in exhibiting the specificity of biological activities. The biosynthesis of the oligosaccharide portion in several ssGPLs is currently being clarified (15, 16, 17, 25, 26), while that of serovar 4-specific GPL is still unresolved. In this study, we have focused on the genomic region predicted to be associated with GPL biosynthesis in the serovar 4 strain and explored the key genes responsible for the formation of 4-O-Me-Rha that might determine the specific properties of MAC serovar 4.

TABLE 1.

Oligosaccharide structures of serovar 2- and 4-specific GPLs