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1.  Development of a Markerless Genetic Exchange Method for Methanosarcina acetivorans C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea 
A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a Δhpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of ΔproC Δhpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of β-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina. A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed ∼300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.
PMCID: PMC368415  PMID: 15006762
2.  Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration 
Applied and Environmental Microbiology  2012;78(22):8112-8121.
Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.
PMCID: PMC3485963  PMID: 22983967
3.  Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1 
Journal of Bacteriology  2002;184(4):1028-1040.
Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1-amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.
PMCID: PMC134814  PMID: 11807063
4.  Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1 
Archaea  2002;1(2):143-150.
The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster in M. mazei Gö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2) located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon.
PMCID: PMC2685556  PMID: 15803652
GlnB-like proteins; nif genes; nitrogen fixation; nitrogen regulation
5.  Markerless Mutagenesis in Methanococcus maripaludis Demonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease 
Journal of Bacteriology  2005;187(3):972-979.
Among the archaea, Methanococcus maripaludis has the unusual ability to use l- or d-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on d-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the l isomer, and the racemase converts the d isomer to the l isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.
PMCID: PMC545699  PMID: 15659675
6.  Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of Nɛ-Acetyl-β-Lysine and Growth at High Salinity 
Applied and Environmental Microbiology  2003;69(10):6047-6055.
The compatible solute Nɛ-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of Nɛ-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB), which are assumed to catalyze Nɛ-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gö1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei Gö1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Δabl mutants of M. maripaludis no longer produced Nɛ-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for Nɛ-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.
PMCID: PMC201229  PMID: 14532061
7.  Development of a Method for Markerless Genetic Exchange in Enterococcus faecalis and Its Use in Construction of a srtA Mutant 
Applied and Environmental Microbiology  2005;71(10):5837-5849.
Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, genetic analysis of these organisms has thus far been limited in scope due to the lack of advanced genetic tools. To broaden the repertoire of genetic tools available for manipulation of E.faecalis, we investigated the use of phosphoribosyl transferases as elements of a counterselection strategy. We report here the development of a counterselectable markerless genetic exchange system based on the upp-encoded uracil phosphoribosyl transferase of E. faecalis. Whereas wild-type E. faecalis is sensitive to growth inhibition by the toxic base analog 5-fluorouracil (5-FU), a mutant bearing an in-frame deletion of upp is resistant to 5-FU. When a cloned version of upp was ectopically introduced into the deletion mutant, sensitivity to 5-FU growth inhibition was restored, thereby providing the basis for a two-step integration and excision strategy for the transfer of mutant alleles to the enterococcal chromosome by recombination. This method was validated by the construction of a ΔsrtA mutant of E. faecalis and by the exchange of the surface protein Asc10, encoded on the pheromone-responsive conjugative plasmid pCF10, with a previously isolated mutant allele. Analysis of the ΔsrtA mutant indicated that SrtA anchors Asc10 to the enterococcal cell wall, facilitating the pheromone-induced aggregation of E. faecalis cells required for high-frequency conjugative plasmid transfer in liquid matings. The system of markerless exchange reported here will facilitate detailed genetic analysis of these important pathogens.
PMCID: PMC1265997  PMID: 16204495
8.  Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿ 
Applied and Environmental Microbiology  2011;77(22):8025-8033.
Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.
PMCID: PMC3208986  PMID: 21948849
9.  Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency▿ † 
Applied and Environmental Microbiology  2009;75(24):7682-7691.
In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3′)-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.
PMCID: PMC2794091  PMID: 19837844
10.  Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus 
In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrF ΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.
PMCID: PMC3993132  PMID: 24487541
11.  Identification of the gene for disaggregatase from Methanosarcina mazei  
Archaea  2008;2(3):185-191.
The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates of Methanosarcina mazei to single cells, were determined for three strains of M. mazei (S-6T, LYC and TMA). The dag genes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genus Methanosarcina and may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed in Escherichia coli and preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed that dag was transcribed to mRNA in M. mazei LYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change of Methanosarcina spp. from aggregate to single cells has been identified.
PMCID: PMC2685598  PMID: 19054745
methanochondroitin; morphological change
12.  An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains 
Nucleic Acids Research  2012;40(21):10964-10979.
We report on the characterization and target analysis of the small (s)RNA162 in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5′ fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA162 (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA162 is crucial for target interactions. Furthermore, our results indicate that sRNA162-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 5′ end of sRNA162 targets the 5′-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA162 acts as an antisense (as)RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.
PMCID: PMC3510493  PMID: 22965121
13.  Allelic Exchange in Actinomyces oris with mCherry Fluorescence Counterselection ▿  
Applied and Environmental Microbiology  2010;76(17):5987-5989.
Described here is a method for facile generation of markerless gene deletion mutants of Actinomyces oris. Homologous integration of a nonreplicative vector carrying a gene exchange cassette into the bacterial chromosome was selected for by using mCherry fluorescence and resistance to kanamycin. Completion of allelic replacement was counterselected for by using loss of fluorescence.
PMCID: PMC2935053  PMID: 20601506
14.  Identification and functional characterization of small non-coding RNAs in Xanthomonas oryzae pathovar oryzae 
BMC Genomics  2011;12:87.
Small non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. Xanthomonas oryzae pathovar oryzae (Xoo) is an important plant pathogenic bacterium which causes serious bacterial blight of rice. However, little is known about the number, genomic distribution and biological functions of sRNAs in Xoo.
Here, we performed a systematic screen to identify sRNAs in the Xoo strain PXO99. A total of 850 putative non-coding RNA sequences originated from intergenic and gene antisense regions were identified by cloning, of which 63 were also identified as sRNA candidates by computational prediction, thus were considered as Xoo sRNA candidates. Northern blot hybridization confirmed the size and expression of 6 sRNA candidates and other 2 cloned small RNA sequences, which were then added to the sRNA candidate list. We further examined the expression profiles of the eight sRNAs in an hfq deletion mutant and found that two of them showed drastically decreased expression levels, and another exhibited an Hfq-dependent transcript processing pattern. Deletion mutants were obtained for seven of the Northern confirmed sRNAs, but none of them exhibited obvious phenotypes. Comparison of the proteomic differences between three of the ΔsRNA mutants and the wild-type strain by two-dimensional gel electrophoresis (2-DE) analysis showed that these sRNAs are involved in multiple physiological and biochemical processes.
We experimentally verified eight sRNAs in a genome-wide screen and uncovered three Hfq-dependent sRNAs in Xoo. Proteomics analysis revealed Xoo sRNAs may take part in various metabolic processes. Taken together, this work represents the first comprehensive screen and functional analysis of sRNAs in rice pathogenic bacteria and facilitates future studies on sRNA-mediated regulatory networks in this important phytopathogen.
PMCID: PMC3039613  PMID: 21276262
15.  Genetic modification of substrate specificity of hypoxanthine phosphoribosyltransferase in Salmonella typhimurium. 
Journal of Bacteriology  1975;121(1):77-82.
Salmonella typhimurium strain GP660 (proAB-gpt deletion, purE) lacks guanine phosphoribosyltransferase and hence cannot utilize guanine as a purine source and is resistant to inhibition by 8-azaguanine. Strain GP660 was mutagenized and a derivative strain (GP36) was isolated for utilization of guanine and hypoxanthine, but not xanthine, as purine sources. This alteration was designated sug. The strain was then sensitive to inhibition by 8-azaguanine. Column chromatographic analysis revealed the altered phosphoribosyltransferase peaks for both hypoxanthine and guanine to be located together, in the same position as hypoxanthine phosphoribosyltransferase (hpt gene product) of the wild-type strain. Genetic analysis showed the sug mutation to be allelic with hpt. Therefore sug represented a modification of the substrate specificity of the hpt gene product.
PMCID: PMC285615  PMID: 1090579
16.  Identification of the Major Expressed S-Layer and Cell Surface-Layer-Related Proteins in the Model Methanogenic Archaea: Methanosarcina barkeri Fusaro and Methanosarcina acetivorans C2A 
Archaea  2012;2012:873589.
Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.
PMCID: PMC3361143  PMID: 22666082
17.  Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae 
PLoS Pathogens  2013;9(1):e1003074.
The strict human pathogen Neisseria gonorrhoeae can utilize homologous recombination to generate antigenic variability in targets of immune surveillance. To evade the host immune response, N. gonorrhoeae promotes high frequency gene conversion events between many silent pilin copies and the expressed pilin locus (pilE), resulting in the production of variant pilin proteins. Previously, we identified a guanine quartet (G4) structure localized near pilE that is required for the homologous recombination reactions leading to pilin antigenic variation (Av). In this work, we demonstrate that inactivating the promoter of a small non-coding RNA (sRNA) that initiates within the G4 forming sequence blocks pilin Av. The sRNA promoter is conserved in all sequenced gonococcal strains, and mutations in the predicted transcript downstream of the G4 forming sequence do not alter pilin Av. A mutation that produces a stronger promoter or substitution of the pilE G4-associated sRNA promoter with a phage promoter (when the phage polymerase was expressed) produced wild-type levels of pilin Av. Altering the direction and orientation of the pilE G4-associated sRNA disrupted pilin Av. In addition, expression of the sRNA at a distal site on the gonococcal chromosome in the context of a promoter mutant did not support pilin Av. We conclude that the DNA containing the G-rich sequence can only form the G4 structure during transcription of this sRNA, thus providing a unique molecular step for the initiation of programmed recombination events.
Author Summary
To evade the host immune response, pathogens have evolved mechanisms to provide genetic diversity in targets of immune surveillance. Organisms that express these diversification systems are under strong evolutionary pressure to provide subpopulations of preexisting variants and often rely on cellular recombination machinery to catalyze dedicated high-frequency reactions without disturbing genome integrity. Previously, we defined a guanine quartet (G4) structure in the strict human pathogen Neisseria gonorrhoeae that is required for initiating the homologous recombination reactions leading to pilin antigenic variation (Av). G4 structures have been implicated in many biological processes, however the mechanisms allowing their formation within a chromosome have not been elucidated. In this work, we show a direct link between transcription of a small RNA (sRNA) that initiates within the G4 structure forming sequence and pilin Av and conclude that the process of transcription is necessary for G4 structure formation. sRNAs have emerged as important regulatory molecules in both eukaryotes and prokaryotes, and this is a novel activity of a sRNA in a bacterium. We anticipate that the reliance of G4 structure formation on transcription is a mechanism used by other biological systems that rely on this alternative DNA structure.
PMCID: PMC3547852  PMID: 23349628
18.  SagS Contributes to the Motile-Sessile Switch and Acts in Concert with BfiSR To Enable Pseudomonas aeruginosa Biofilm Formation▿† 
Journal of Bacteriology  2011;193(23):6614-6628.
The interaction of Pseudomonas aeruginosa with surfaces has been described as a two-stage process requiring distinct signaling events and the reciprocal modulation of small RNAs (sRNAs). However, little is known regarding the relationship between sRNA-modulating pathways active under planktonic or surface-associated growth conditions. Here, we demonstrate that SagS (PA2824), the cognate sensor of HptB, links sRNA-modulating activities via the Gac/HptB/Rsm system postattachment to the signal transduction network BfiSR, previously demonstrated to be required for the development of P. aeruginosa. Consistent with the role of SagS in the GacA-dependent HtpB signaling pathway, inactivation of sagS resulted in hyperattachment, an HptB-dependent increase in rsmYZ, increased Psl polysaccharide production, and increased virulence. Moreover, sagS inactivation rescued attachment but abrogated biofilm formation by the ΔgacA and ΔhptB mutant strains. The ΔsagS strain was impaired in biofilm formation at a stage similar to that of the previously described two-component system BfiSR. Expression of bfiR but not bfiS restored ΔsagS biofilm formation independently of rsmYZ. We demonstrate that SagS interacts directly with BfiS and only indirectly with BfiR, with the direct and specific interaction between these two membrane-bound sensors resulting in the modulation of the phosphorylation state of BfiS in a growth-mode-dependent manner. SagS plays an important role in P. aeruginosa virulence in a manner opposite to that of BfiS. Our findings indicate that SagS acts as a switch by linking the GacA-dependent sensory system under planktonic conditions to the suppression of sRNAs postattachment and to BfiSR, required for the development of P. aeruginosa biofilms, in a sequential and stage-specific manner.
PMCID: PMC3232883  PMID: 21949078
19.  A Novel Inducible Protein Production System and Neomycin Resistance as Selection Marker for Methanosarcina mazei 
Archaea  2012;2012:973743.
Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.
PMCID: PMC3407599  PMID: 22851906
20.  Improved and Versatile Transformation System Allowing Multiple Genetic Manipulations of the Hyperthermophilic Archaeon Thermococcus kodakaraensis 
We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (ΔpyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (ΔpyrF ΔtrpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (ΔpyrF ΔtrpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (ΔpyrF ΔtrpE ΔhisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.
PMCID: PMC1169065  PMID: 16000802
21.  Regulatory RNAs and the HptB/RetS signalling pathways fine-tune Pseudomonas aeruginosa pathogenesis 
Molecular Microbiology  2010;76(6):1427-1443.
Bacterial pathogenesis often depends on regulatory networks, two-component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine-tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti-anti-σ factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS-dependent but HptB-independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine-tuned regulation of gene expression.
PMCID: PMC2904497  PMID: 20398205
22.  A Genome-Wide Approach to Discovery of Small RNAs Involved in Regulation of Virulence in Vibrio cholerae 
PLoS Pathogens  2011;7(7):e1002126.
Small RNAs (sRNAs) are becoming increasingly recognized as important regulators in bacteria. To investigate the contribution of sRNA mediated regulation to virulence in Vibrio cholerae, we performed high throughput sequencing of cDNA generated from sRNA transcripts isolated from a strain ectopically expressing ToxT, the major transcriptional regulator within the virulence gene regulon. We compared this data set with ToxT binding sites determined by pulldown and deep sequencing to identify sRNA promoters directly controlled by ToxT. Analysis of the resulting transcripts with ToxT binding sites in cis revealed two sRNAs within the Vibrio Pathogenicity Island. When deletions of these sRNAs were made and the resulting strains were competed against the parental strain in the infant mouse model of V. cholerae colonization, one, TarB, displayed a variable colonization phenotype dependent on its physiological state at the time of inoculation. We identified a target of TarB as the mRNA for the secreted colonization factor, TcpF. We verified negative regulation of TcpF expression by TarB and, using point mutations that disrupted interaction between TarB and tpcF mRNA, showed that loss of this negative regulation was primarily responsible for the colonization phenotype observed in the TarB deletion mutant.
Author Summary
Vibrio cholerae is the causative agent of the diarrheal disease cholera, which remains a significant public health issue in Africa, South Asia and recently Haiti. To better understand virulence gene regulation in V. cholerae we sought to investigate the contribution of small non-coding regulatory RNAs (sRNAs) to regulation of virulence in V. cholerae. We undertook a genome wide approach to sRNA discovery combining direct sequencing of sRNA transcript cDNA and genome wide binding studies of the master protein regulator of virulence, ToxT. This approach yielded one previously known and 17 new potential sRNAs under the control of ToxT. We investigated one of these new sRNAs and showed that it negatively regulates expression of the secreted colonization factor TcpF, adding a new facet to the complex gene regulatory network necessary for virulence in V. cholerae.
PMCID: PMC3136459  PMID: 21779167
23.  Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays 
BMC Genomics  2012;13:550.
Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49), employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases.
We identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5′ rapid amplification of cDNA ends-PCR (RACE-PCR) analysis.
In accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs appears to be strain specific.
PMCID: PMC3542284  PMID: 23062031
Streptococcus pyogenes; Small noncoding RNAs; Virulence; Transcriptional regulation; Pathogenesis
24.  Purine Salvage Pathways among Borrelia Species▿  
Infection and Immunity  2007;75(8):3877-3884.
Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood.
PMCID: PMC1952022  PMID: 17502392
25.  Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15 
Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5′ UTR was present in the pta-ackA transcript. Removal of the 5′ UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5′ UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15.
PMCID: PMC3911069  PMID: 24317083

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