Advances in Development of a Genetic System forThermoanaerobacterium spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

ABSTRACT Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system,Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticumJW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of theThermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.

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Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

Journal of Bacteriology ◽

10.1128/jb.181.13.3981-3993.1999 ◽

1999 ◽

Vol 181 (13) ◽

pp. 3981-3993 ◽

Cited By ~ 195

Author(s):

Sylvia A. Denome ◽

Pamela K. Elf ◽

Thomas A. Henderson ◽

David E. Nelson ◽

Kevin D. Young

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Binding Proteins ◽

Kanamycin Resistance ◽

Open Reading Frames ◽

Penicillin Binding Proteins ◽

E Coli ◽

Peptidoglycan Biosynthesis ◽

Kanamycin Resistance Cassette ◽

Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

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Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01382-07 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5411-5420 ◽

Cited By ~ 22

Author(s):

Yu-Sin Jang ◽

Young Ryul Jung ◽

Sang Yup Lee ◽

Ji Mahn Kim ◽

Jeong Wook Lee ◽

...

Keyword(s):

Succinic Acid ◽

Shuttle Vector ◽

Green Fluorescence Protein ◽

Expression Vectors ◽

Rumen Bacteria ◽

Gene Encoding ◽

Shuttle Vectors ◽

Genes Encoding ◽

Fluorescence Protein

ABSTRACT Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 106 and 7.1 × 106 transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

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Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola

Applied and Environmental Microbiology ◽

10.1128/aem.00478-15 ◽

2015 ◽

Vol 81 (13) ◽

pp. 4329-4338 ◽

Cited By ~ 7

Author(s):

Yuebin Li ◽

John Ruby ◽

Hui Wu

Keyword(s):

Genetic Manipulation ◽

Periodontal Diseases ◽

Genetic System ◽

Kanamycin Resistance ◽

Treponema Denticola ◽

Oral Pathogen ◽

Content Type ◽

Kanamycin Resistance Cassette ◽

Allelic Replacement ◽

Kanamycin Cassette

ABSTRACTTreponema denticolahas been recognized as an important oral pathogen of the “red complex” bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence ofT. denticoladue to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation ofT. denticolaATCC 35405. Compared to the widely usedermF-ermAMcassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistantT. denticolacolonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames ofT. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional intrans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had theflgEgene coding for PF hook protein inactivated. The integration of the full-lengthflgEgene into the genome of theflgEmutant rescued all of the defects associated with theflgEmutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation ofT. denticolathat will facilitate the characterization of virulence factors attributed to this important oral pathogen.

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Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

Infection and Immunity ◽

10.1128/iai.71.6.3320-3328.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3320-3328 ◽

Cited By ~ 26

Author(s):

Aloka B. Bandara ◽

Mark L. Lawrence ◽

Hugo P. Veit ◽

Thomas J. Inzana

Keyword(s):

Parent Strain ◽

Capsular Polysaccharide ◽

Shuttle Vector ◽

Actinobacillus Pleuropneumoniae ◽

Kanamycin Resistance ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Homology ◽

Suicide Vector ◽

Kanamycin Resistance Cassette ◽

Serotype 1

ABSTRACT The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kanr) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kanr gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Δcps1N and strain 4074Δcps1B, respectively. Strain 4074Δcps1N produced no detectable CP, but strain 4074Δcps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Δcps1N to produce 4074Δcps1N(pABcps101), 4074Δcps1N(pJMLcps53), and 4074Δcps1N(pABcps55), respectively. Strain 4074Δcps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Δcps1N(pJMLcps53) and 4074Δcps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Δcps1N(pABcps101) ≥ strain 4074Δcps1N > strain 4074Δcps1B. Strain 4074Δcps1N(pJMLcps53) was less virulent than strain 4074Δcps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

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Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori

Applied and Environmental Microbiology ◽

10.1128/aem.01348-07 ◽

2008 ◽

Vol 74 (7) ◽

pp. 2095-2102 ◽

Cited By ~ 47

Author(s):

Ivo G. Boneca ◽

Chantal Ecobichon ◽

Catherine Chaput ◽

Aurélie Mathieu ◽

Stéphanie Guadagnini ◽

...

Keyword(s):

Helicobacter Pylori ◽

Shuttle Vector ◽

Expression System ◽

Gene Promoter ◽

Start Codon ◽

Expression Of Genes ◽

Genes Encoding ◽

Kanamycin Cassette ◽

H Pylori

ABSTRACT The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.

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Isolation and Characterization of a Native Composite Transposon, Tn14751, Carrying 17.4 Kilobases of Corynebacterium glutamicum Chromosomal DNA

Applied and Environmental Microbiology ◽

10.1128/aem.71.1.407-416.2005 ◽

2005 ◽

Vol 71 (1) ◽

pp. 407-416 ◽

Cited By ~ 10

Author(s):

Masayuki Inui ◽

Yota Tsuge ◽

Nobuaki Suzuki ◽

Alain A. VertÃ¯Â¿Â½s ◽

Hideaki Yukawa

Keyword(s):

Corynebacterium Glutamicum ◽

Insertion Sequence ◽

Kanamycin Resistance ◽

Chromosomal Dna ◽

Kanamycin Resistance Cassette ◽

Isolation And Characterization ◽

Genes Encoding ◽

Genome Scale ◽

Random Insertion

ABSTRACT A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 Ã¯Â¿Â½ 102 transformants per μg of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

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Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon Pyrococcus abyssi

Applied and Environmental Microbiology ◽

10.1128/aem.68.11.5528-5536.2002 ◽

2002 ◽

Vol 68 (11) ◽

pp. 5528-5536 ◽

Cited By ~ 53

Author(s):

Soizick Lucas ◽

Laurent Toffin ◽

Yvan Zivanovic ◽

Daniel Charlier ◽

Hélène Moussard ◽

...

Keyword(s):

Shuttle Vector ◽

Low Frequency ◽

Vector System ◽

Hyperthermophilic Archaea ◽

Gene Encoding ◽

Hyperthermophilic Archaeon ◽

Shuttle Vectors ◽

Bacterial Vector ◽

Genes Encoding ◽

Pyrococcus Abyssi

ABSTRACT Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (102 to 103 transformants per μg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

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ς54 Promoters Control Expression of Genes Encoding the Hook and Basal Body Complex in Rhodobacter sphaeroides

Journal of Bacteriology ◽

10.1128/jb.182.20.5787-5792.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5787-5792 ◽

Cited By ~ 14

Author(s):

Sebastian Poggio ◽

Carlos Aguilar ◽

Aurora Osorio ◽

Bertha González-Pedrajo ◽

Georges Dreyfus ◽

...

Keyword(s):

Gene Expression ◽

Rhodobacter Sphaeroides ◽

Basal Body ◽

Genetic System ◽

Site Directed Mutagenesis ◽

Expression Of Genes ◽

Intracellular Signals ◽

Serovar Typhimurium ◽

Genes Encoding ◽

External Stimuli

ABSTRACT Gene expression of the flagellar system is tightly controlled by external stimuli or intracellular signals. A general picture of this regulation has been obtained from studies of Salmonella enterica serovar Typhimurium. However, these regulatory mechanisms do not apply to all bacterial groups. In this study, we have investigated regulation of the flagellar genetic system inRhodobacter sphaeroides. Deletion analysis, site-directed mutagenesis, and 5′-end mapping were conducted in order to identify the fliO promoter. Our results indicate that this promoter is recognized by the factor ς54. Additionally, 5′-end mapping of the flgB andfliK transcripts suggests that these mRNAs are also transcribed from ς54 promoters. Finally, we showed evidence that suggests that fliC transcription is not entirely dependent on the presence of a complete basal body-hook structure. Our results are discussed in the context of a possible regulatory hierarchy controlling flagellar gene expression in R. sphaeroides.

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Expression of bacteriophage ϕEa1h lysozyme in Escherichia coli and its activity in growth inhibition of Erwinia amylovora

Microbiology ◽

10.1099/mic.0.27224-0 ◽

2004 ◽

Vol 150 (8) ◽

pp. 2707-2714 ◽

Cited By ~ 33

Author(s):

Won-Sik Kim ◽

Heike Salm ◽

Klaus Geider

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Expression Vector ◽

Erwinia Amylovora ◽

Kanamycin Resistance ◽

Target Cells ◽

Sequence Alignments ◽

E Coli ◽

Kanamycin Resistance Cassette ◽

Genes Encoding

A 3·3 kb fragment from Erwinia amylovora phage ϕEa1h in plasmid pJH94 was previously characterized and found to contain an exopolysaccharide depolymerase (dpo) gene and two additional ORFs encoding 178 and 119 amino acids. ORF178 (lyz) and ORF119 (hol) were found to overlap by 19 bp and they resembled genes encoding lysozymes and holins. In nucleotide sequence alignments, lyz had structurally conserved regions with residues important for lysozyme function. The lyz gene was cloned into an expression vector and expressed in Escherichia coli. Active lysozyme was detected only when E. coli cells with the lyz gene and a kanamycin-resistance cassette were grown in the presence of kanamycin. Growth of Erw. amylovora was inhibited after addition of enzyme exceeding a threshold for lysozyme to target cells. When immature pears were soaked in lysates of induced cells, symptoms such as ooze formation and necrosis were retarded or inhibited after inoculation with Erw. amylovora.

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