A Mycobacterial Systems Resource for the Research Community.

Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulceransIMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.

Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages.

Effective chemotherapy of tuberculosis requires rapid assessment of drug sensitivity because of the emergence of multidrug-resistant Mycobacterium tuberculosis. Drug susceptibility was assessed by a simple method based on the efficient production of photons by viable mycobacteria infected with specific reporter phages expressing the firefly luciferase gene. Light production was dependent on phage infection, expression of the luciferase gene, and the level of cellular adenosine triphosphate. Signals could be detected within minutes after infection of virulent M. tuberculosis with reporter phages. Culture of conventional strains with antituberculosis drugs, including isoniazid or rifampicin, resulted in extinction of light production. In contrast, light signals after luciferase reporter phage infection of drug-resistant strains continued to be produced. Luciferase reporter phages may help to reduce the time required for establishing antibiotic sensitivity of M. tuberculosis strains from weeks to days and to accelerate screening for new antituberculosis drugs.

Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins.

Similarity between the DNA substrates and products of integrase-mediated site-specific recombination reactions results in a single recombinase enzyme being able to catalyze both the integration and excision reactions. The control of directionality in these reactions is achieved through a class of small accessory factors that favor one reaction while interfering with the other. These proteins, which we will refer to collectively as recombination directionality factors (RDFs), play architectural roles in reactions catalyzed by their cognate recombinases and have been identified in conjunction with both tyrosine and serine integrases. Previously identified RDFs are typically small, basic and have diverse amino acid sequences. A subset of RDFs, the cox genes, also function as transcriptional regulators. We present here a compilation of all the known RDF proteins as well as those identified through database mining that we predict to be involved in conferring recombination directionality. Analysis of this group of proteins shows that they can be grouped into distinct sub-groups based on their sequence similarities and that they are likely to have arisen from several independent evolutionary lineages. This compilation will prove useful in recognizing new proteins that confer directionality upon site-specific recombination reactions encoded by plasmids, transposons, phages and prophages.

Genetic transformation of mycobacteria.

Recent advances in methods for introducing DNA into both fast- and slow-growing species of mycobacteria have stimulated the construction of a variety of cloning vectors and the development of a versatile genetic system. These tools will facilitate studies of mycobacterial pathogenicity, antibiotic action and drug resistance, and lead to more effective methods for diagnosis, prevention and treatment of mycobacterial diseases.

The origins and ongoing evolution of viruses.

Genome analyses of double strand DNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution.

Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages.

We report the complete genome DNA sequences of HK97 (39,732 bp) and HK022 (40,751 bp), double-stranded DNA bacteriophages of Escherichia coli and members of the lambdoid or lambda-like group of phages. We provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of E. coli phage lambda and Salmonella typhimurium phage P22. The comparisons confirm that these phages are genetic mosaics, with mosaic segments separated by sharp transitions in the sequence. The mosaicism provides clear evidence that horizontal exchange of genetic material is a major component of evolution for these viruses. The data suggest a model for evolution in which diversity is generated by a combination of illegitimate and homologous recombination and mutational drift, and selection for function produces a population in which most of the surviving mosaic boundaries are located at gene boundaries or, in some cases, at protein domain boundaries within genes. Comparisons of these genomes highlight a number of differences that allow plausible inferences of specific evolutionary scenarios for some parts of the genome. The comparative analysis also allows some inferences about function of genes or other genetic elements. We give examples for the generalized recombination genes of HK97, HK022 and P22, and for a putative headtail adaptor protein of HK97 and HK022. We also use the comparative approach to identify a new class of genetic elements, the morons, which consist of a protein-coding region flanked by a putative delta 70 promoter and a putative factor-independent transcription terminator, all located between two genes that may be adjacent in a different phage. We argue that morons are autonomous genetic modules that are expressed from the repressed prophage. Sequence composition of the morons implies that they have entered the phages' genomes by horizontal transfer in relatively recent evolutionary time.

Genomic sequence and analysis of the atypical temperate bacteriophage N15.

N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. Here, we describe the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.

Genome structure of mycobacteriophage D29: implications for phage evolution.

Mycobacteriophage D29 is a lytic phage that infects both fast and slow-growing mycobacterial species. The complete genome sequence of D29 reveals that it is a close relative of the temperate mycobacteriophage L5, whose sequence has been described previously. The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens. Comparison of the two genomes shows that they are punctuated by a large number of insertions, deletions, and substitutions of genes, consistent with the genetic mosaicism of lambdoid phages.

Characterization of the mycobacteriophage L5 attachment site, attP.

Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.

Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into gamma delta resolvase.

The ability to determine protein structures by X-ray crystallography is often thwarted by the difficulty of finding isomorphous heavy-atom derivatives. The crystal structure of the site-specific recombinase, resolvase, has been difficult to determine for this reason. We have overcome this problem by introducing 13 single cysteine substitutions into the resolvase catalytic domain using oligonucleotide mutagenesis. The mutant proteins were screened for their ability to crystallize into the orthorhombic form and bind mercury ions isomorphously. Two mutant proteins provided excellent heavy-atom derivatives. This approach should be of general use and particularly helpful in cases where traditional methods have failed to produce a derivative.

A putative ABC-transport operon of Mycobacterium smegmatis.

We have recently described the mpr gene of Mycobacterium smegmatis whose product confers resistance to mycobacteriophages L5 and D29 when overproduced (Barsom and Hatfull (1996) Mol. Microbiol. 21, 159-170). We have determined the nt sequence of approximately 3.5 kb immediately adjacent to mpr which appears to encode components of an ATP-binding cassette (ABC) transport system. Four closely-spaced open reading frames (ORF) were identified although two of these may cooperate to produce an integral membrane component of the transport system via a programmed translational frameshift. Another putative protein is also predicted to be an integral membrane protein, while the third is an ABC-transporter protein. We propose that these three putative proteins form a mycobacterial membrane-bound complex involved in protein-dependent transport. This is the first ABC-transport system to be described in mycobacteria.

Mycobacteriophage D29 integrase-mediated recombination: specificity of mycobacteriophage integration.

Mycobacteriophage D29 is a lytic phage that infects both fast- and slow-growing species of the mycobacteria. D29 forms clear plaques on lawns of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guérin (BCG) in which a very high proportion of infected cells are killed. However, genomic analysis of D29 demonstrates that it is a close relative of the temperate mycobacteriophage L5, and is presumably a non-temperate derivative of a temperate parent. The D29 genome encodes a putative integrase protein with a primary amino acid sequence similar to that of the L5 integrase; the corresponding int genes fall in colinear positions within the D29 and L5 genomes, immediately flanking and transcribed away from their associated attP sites. We show here that the D29 integrase is functional and catalyzes integrative recombination between the D29 attP site and the M. smegmatis attB site in vitro in an mIHF-dependent manner. D29 integrase also mediates recombination between the L5 attP site and attB DNA and, reciprocally, L5 integrase catalyzes recombination with D29 attP DNA. However, in both in-vitro and in-vivo assays, the D29-encoded integrase recombines the D29 attP more efficiently than the L5 attP, and vice versa, suggesting that each integration system has evolved a degree of specificity of attP recognition. We also present the sequences of the putative attP site and integrase protein of the cryptic prophage-like element phiRv2, and compare them to those of mycobacteriophages L5 and D29.

Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria.

Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.

Transcriptional regulation and immunity in mycobacteriophage Bxb1.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis that shares a similar genome organization to mycobacteriophage L5, although the two phages are heteroimmune. We have investigated the regulatory circuitry of Bxb1 and found that it encodes a repressor, gp69, which regulates at least two promoters, an early lytic promoter, Pleft, and the divergent promoter, Pright. Bxb1 gp69 is 41% identical to the L5 repressor (gp71) and binds to repressor binding sites that conform to a similar, but distinct, 13 bp asymmetric consensus sequence to that for the L5 gp71 binding sites. The two phage repressors have a strong preference for their cognate binding sites, thus accounting for their immunity phenotypes. The Bxb1 genome contains 34 putative repressor binding sites located throughout the genome, but situated within short intergenic spaces and orientated in only one direction relative to the direction of transcription. Comparison with the locations of repressor binding sites within the L5 genome provides insights into how these unusual regulatory systems evolve.

Mycobacteriophage L5 integrase-mediated site-specific integration in vitro.

Mycobacteriophage L5, a temperate phage of the mycobacteria, forms stable lysogens in Mycobacterium smegmatis via site-specific integration of the phage genome. Recombination occurs within specific phage and bacterial attachment sites and is catalyzed by the phage-encoded integrase protein in vivo. We describe here the overexpression and purification of L5 integrase and its ability to mediate integrative recombination in vitro. We find that L5 integrase-mediated recombination is greatly stimulated by extracts of M. smegmatis but not by Escherichia coli extracts, purified E. coli integration host factor, or purified HU, indicating the presence of a novel mycobacterial integration host factor.

Deletion of the spf (spot 42 RNA) gene of Escherichia coli.

To investigate the function of spot 42 RNA, a small RNA of Escherichia coli, we constructed a strain in which spf, the structural gene for this RNA, is deleted. We achieved this by using a delta att phage lambda carrying a DNA fragment spanning the spf region but with a precise deletion of spf. By integration of this phage at the spf locus and by its subsequent excision, we were able to cross the spf deletion onto the bacterial chromosome. The fact that such a deletion could be obtained indicated that spf is not an essential gene. We did not observe any major defect in delta spf cells, although in one strain background the deletion caused a slight growth impairment.