Veterinary Microbiology 173 (2014) 371–378

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Short communication

Natural competence in Histophilus somni strain 2336 Nehal Shah a, Aloka B. Bandara a, Indra Sandal a,1, Thomas J. Inzana a,b,* a b

Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061, USA Virginia Tech Carilion School of Medicine, Virginia Tech, Blacksburg, VA 24061, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 January 2014 Received in revised form 9 July 2014 Accepted 26 July 2014

Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases of bovines. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. However, to identify the genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult, likely due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and shows a strong bias for fragments containing specific uptake signal sequences (USS) within the genome. We hypothesized that natural transformation may also be possible in H. somni strain 2336 because its genome is over-represented with H. influenzae USS (50 -AAGTGCGGT-30 ) and contains most of the genes necessary for competence. H. somni strain 2336 was successfully transformed and mutated with genomic linear DNA from an H. somni mutant (738Dlob2a), which contains a kanamycinresistance (KanR) gene and the USS within lob2A. Although most of the competence genes found in H. influenzae were present in H. somni, comD and the 50 portion of comE were absent, which may account for the low transformation efficiency. The transformation efficiency of strain 2336 was greatest during mid-log growth phase and when cyclic adenosine monophosphate was added to the transformation medium. However, mutants were not isolated when strain 2336 was transformed with genomic DNA containing the same KanR gene from H. somni luxS or uspE mutants, which lack the USS in these specific genes. Shuttle vector pNS3K was also naturally transformed into strain 2336, though at a lower efficiency. However, natural transformation with either H. somni linear DNA (2336Dlob2A) or pNS3K was unsuccessful with H. somni commensal strain 129Pt and several other disease isolates. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Histophilus somni Competence Transformation Mutagenesis Shuttle vector

1. Introduction

* Corresponding author at: Life Sciences 1, 970 Washington St., SW, Virginia Tech, Blacksburg, VA 24061, USA. Tel.: +1 540 231 5188; fax: +1 540 231 5553. E-mail addresses: [email protected], [email protected] (T.J. Inzana). 1 Current address: Department of Internal Medicine, Division of Pulmonary Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132, USA. http://dx.doi.org/10.1016/j.vetmic.2014.07.025 0378-1135/ß 2014 Elsevier B.V. All rights reserved.

Histophilus somni is an opportunistic pathogen of cattle and one of the primary agents involved in bovine respiratory disease. H. somni is also responsible for a wide variety of systemic infections in cattle, including myocarditis, arthritis, thrombotic meningoencephalitis (TME), and others (Siddaramppa and Inzana, 2004). Although closely related to Haemophilus influenzae, mutagenesis and expression of foreign DNA in H. somni is difficult due to the bacterium’s apparently tight

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restriction-modification systems (Sandal et al., 2008; Siddaramappa et al., 2011). In order to identify genes involved in virulence and further understand the bacterium’s pathogenic mechanisms, isogenic mutants need to be generated. There have only been four genes disrupted by site-specific mutagenesis in H. somni: lob2A (Wu et al., 2000), p76 (Sanders et al., 2003), aroA (Briggs and Tatum, 2005), and ibpA (Hoshinoo et al., 2009). Bacteria that are naturally competent are able to actively take up DNA from their extracellular environment. Furthermore, if there is sufficient similarity, these sequences may recombine within the host genome (Maughan et al., 2008). Mutagenesis using natural transformation is well established in H. influenzae. Once competence is induced, genes homologous to components of the Type IV pilus machinery and of the ComEC/Rec2 membrane channel (Chen and Dubnau, 2004) are expressed and are responsible for binding and bringing extracellular DNA across the outer membrane into the periplasm. Uptake of extracellular DNA is strongly biased for fragments containing a specific uptake signal sequence (USS) motif, which has been identified in H. influenzae as 50 -AAGTGCGGT-30 (Redfield et al., 2006). These USS motifs have also been found to be overrepresented in the genome of H. somni strains 129Pt and 2336 (Challacombe et al., 2007). Therefore, we sought to determine if H. somni could be made competent to take up DNA through natural transformation, which could improve genetic manipulation of this important pathogen. 2. Materials and methods 2.1. Bacterial strains and growth conditions The bacterial strains used in this study are listed in Table 1. Escherichia coli was grown in Luria–Bertani (LB) broth or on LB agar (BD, San Jose, CA) at 37 8C. Antibiotics for selection were used at the following concentrations: kanamycin, 50 mg/ml; streptomycin, 80 mg/ml; ampicillin, 50 mg/ml. H. somni strains were grown on Columbia Blood Agar (CBA) plates or BBL Brain Heart Infusion (BHI) agar plates (BD), both supplemented with 5% sheep blood. Plates were incubated overnight at 37 8C in 5% CO2. For

natural transformation broth cultures were grown in Bacto BHI broth (BD) supplemented with 0.1% Trizma Base and 0.01% thiamine monophosphate (TMP) (BHI-TT) (Sigma Chemical Co., St. Louis, MO) (Inzana and Corbeil, 1987). Broth cultures were shaken at 200 rpm at 37 8C. Kanamycin was used at 80 mg/ml for selection of H. somni recombinant clones by natural transformation, and for clones containing plasmid DNA. The M-IV medium for natural transformation of H. influenzae was prepared as described (Poje and Redfield, 2003). 2.2. Natural transformation The protocol for natural transformation was modified from Poje and Redfield (2003). H. somni strain 2336 was grown to log phase (about 3.2  109 colony forming units [CFU]/ml), harvested at 4350  g for 10 min at room temperature, washed twice in M-IV medium, suspended in the original volume of M-IV medium, and incubated for 100 min at 100 rpm at 37 8C to induce competence. One-ml volumes of competent cells were incubated with 1 mg of homologous genomic DNA isolated from a lob2A mutant of H. somni strain 738 (738Dlob2A) (Wu et al., 2000), or from strain 2336 luxS or uspE mutants obtained by Tn mutagenesis, both of which contained the same Tn5 kanamycin resistance cassette (KanR) as 738Dlob2A. The bacteria were incubated at 37 8C for 30 min. and half of the suspension was spread onto CBA with kanamycin (80 mg/ml). The transformation efficiency of strain 2336 at different growth stages was evaluated by growing the bacteria in BHI-TT to a Klett unit optical density of 60 (lag phase), 90 (early log phase), 120 (mid-log phase), or 180 (stationary phase). The cells were harvested, washed and incubated in M-IV medium, and transformed with 1.0 mg of 738Dlob2A DNA, and cultured, as described above. In addition, the effect of cyclic adenosine monophosphate (cAMP) supplementation on the transformation efficiency of H. somni grown to mid-log phase was tested. Strains 129Pt and 2336 were grown in BHI-TT, harvested, and incubated for 100 min in M-IV medium supplemented with or without 2 mM cAMP. The cells were subsequently incubated with 1 mg of genomic DNA from strain 738Dlob2A DNA, and cultured on CBA containing 80 mg/ml kanamycin.

Table 1 Bacterial strains and plasmids used in this study. Bacterium

Strain

Description

Source or reference

H. somni strains

2336 129pt M14-622 649 8025

Pneumonia isolate Isolate from the prepuce of a healthy bull Pneumonia isolate from local herd Abortion isolate Thrombotic meningoencephalitis Isolate Pneumonia isolate Strain 738 containing a kanamycin resistance marker within lob2A by allelic exchange Strain 2336 containing a kanamycin resistance marker within luxS by transposon mutagenesis Strain 2336 containing a kanamycin resistance marker within uspE by transposon mutagenesis Broad host-range shuttle vector High-efficiency H. somni shuttle vector

Corbeil et al. (1985) Corbeil et al. (1985) This work Corbeil et al. (1985)

7735 738Dlob2A 2336DluxS 2336DuspE Plasmids

pLS88 pNS3K

Corbeil et al. (1985) Elswaifi et al. (2009) Wu et al. (2000) This work This work Willson et al. (1989) Sandal et al. (2008)

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Table 2 Primers used in this work. Primer

Description

Sequence

lob2A F lob2A R uspE F uspE R luxS F luxS R LuxS_Forw LuxS_Rev comD F comD R comE F comE R KanF KanR

lob2A forward primer lob2A reverse primer uspE forward primer uspE reverse primer luxS forward primer luxS reverse primer Alternative luxS forward primer Alternative luxS reverse primer comD forward primer comD reverse primer comE forward primer comE reverse primer Kanamycin Tn5 forward primer Kanamycin Tn5 reverse primer

50 -AGCAATCTCGCAAATAAA-30 (Wu et al., 2000) 50 -ATCTTTCAAATATCCTTCTTCTA-30 (Wu et al., 2000) 50 -ATGCAATTTAATAATATACTTGTCGTTTT-30 50 -TTATTTTTTACTCGGTTTAATTGCCAAC-30 50 -ACAATGTCATGACCTGCTCGAT-30 50 -CACAAGGAATGCCAAGGTTTTC-30 50 -CGGGGATCCTTGTTATAGCAATACGTTACAATAC-30 50 -CGGTCTAGAAAGTGCGGTTTAAAGCTGTTCCTAATATTGTCAT-30 50 -ATGAAACATTGGTTTTTCCTGATT-30 50 -CCATTCACTACTATCACATTG-30 50 -ATTTTTTAGTATGTTTTTGTTTGCC-30 50 -TTATTTTTTACCCTCACTTTTTTGTTT-30 50 -AGAGGCTATTCGGCTATGACTGGGCA-30 50 -CGGTCCGCCACACCCAGCCGGCCACAG-30

In addition, the natural transformation efficiency of H. somni strains 649 (abortion isolate), 7735 (pneumonia isolate), 8025 (TME isolate), and M14-622 (a local recent pneumonia isolate) was also examined. The strains were grown to approximately 8.5  109 CFU/ml in BHI-TT and incubated in M-IV medium supplemented with 2 mM cAMP, as described above. The presence of the lob2A and luxS genes in these strains was examined by PCR (see Table 2 for primers). For transformation of circular plasmids, 3.2  109 CFU of cells that were washed and resuspended in 1.0 ml of MIV medium were added to 300 ml of 80% glycerol (added to induce osmotic shock and promote uptake across the inner membrane (Poje and Redfield, 2003) and 0.5 mg of plasmid pNS3K (Sandal et al., 2008) and the mixture incubated at 37 8C for 30 min. The bacteria were sedimented at 9000  g for 1 min, and 1 ml of supernatant removed. The bacteria were resuspended in the remaining suspension and spread onto CBA containing kanamycin (80 mg/ml). Kanamycin-resistant colonies were streaked onto fresh plates and blotted onto Nylon membranes. Colonies containing KanR were identified by hybridization with an oligonucleotide probe, which was synthesized using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science, Branford, CT). DNA hybridization and colony detection were performed using the same kit, following the manufacturer’s recommendations. The presence of KanR was used to differentiate spontaneous kanamycin-resistant colonies from double-crossover mutants (Green, 2012). The presence of the KanR gene and lack of lob2A were confirmed by PCR (Fig. 1). The confirmed recombinant clones were designated 2336Dlob2A.

Kanamycin-resistant colonies for plasmid transformation were streaked onto CBA plates with kanamycin (80 mg/ ml). The presence of plasmids was determined by resuspending 1 loopful of bacteria in 50 ml of water, followed by addition of 50 ml of phenol:chloroform:isoamyl alcohol, and vortexing for 10 s. The cells were sedimented at 9000  g at 4 8C and 30 ml of the aqueous phase was analyzed by gel electrophoresis. Alternatively, plasmids were isolated using QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA), followed by gel electrophoresis. Transformation frequencies were calculated by dividing the number of transformants/ml by the number of viable cells/ml (Maughan and Redfield, 2009). 2.3. Molecular methods DNA Taq Polymerase, restriction enzymes, and their associated buffers were purchased from New England Biolabs (NEB, Beverly, MA). Genomic DNA (gDNA) was extracted using the DNeasy1 Blood and Tissue Kit (Qiagen, Valencia, CA). Plasmids were isolated using the Miniprep Spin Kit (Qiagen). Polymerase chain reactions (PCR) were carried out in 25 ml, and consisted of 2.5 ml of 10 Taq reaction buffer (NEB), 200 nM of primers, 1 mM MgCl2, and 60 ng of plasmid template DNA, 100 ng of chromosomal template DNA, or 1 ml of boiled cell extract containing gDNA. For the latter, a small number of colonies were suspended in 100 ml of water, boiled at 100 8C for 10 min, and unlysed cells sedimented at 9300  g for 10 min at 4 8C. The supernatant was used as a template for PCR. The PCR primers used were designed based on the genome sequence of

Fig. 1. Diagram of the lob2A loci from wild type and a KanR mutant showing the positions of the USS motif (USS) (50 -AAGTGCGGT-30 ) and the lob2A forward and reverse primers (lob2AF and lob2AR, respectively) and the KanR forward and reverse primers (KanF and KanR), respectively. Numbers refer to nucleotide base pairs of the sequences shown.

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H. influenzae strain KW20 (accession # L42023), and are shown in Table 2. DNA was heated to 94 8C for 5 min for initial denaturation, followed by 32 cycles of a three-temperature cycling protocol (94 8C for 1 min for denaturation, 2 min for annealing, and 72 8C for 1 min for extension) and one cycle at 72 8C for 10 min for final extension. The annealing temperature varied depending on the primers used, and was always 2–3 8C below their melting temperatures. 2.4. Transposon mutagenesis Transposon (Tn) mutagenesis using the EZ::Tn5TM(KAN-2)Tnp Transposome (Epicentre, Madison, WI) was carried out as described by Sandal et al. (Sandal et al., 2011). The Tn insertion sites were amplified by random amplification of Tn ends, and identified by sequencing one end of the Tn and a region of flanking chromosomal DNA (Ducey and Dyer, 2002). The Tn mutants with KanR insertions in uspE or luxS were screened for deficiency in biofilm formation in tubes (Sandal et al., 2007), and loss of virulence in mice (Wu et al., 2000). Mutant 2336DuspE was highly deficient in biofilm formation, while both mutants 2336DuspE and 2336DluxS were highly attenuated in a mouse model of acute septicemia (unpublished data). 2.5. Bioinformatic analysis of H. somni com genes and encoded proteins The genomic sequences of H. somni strains 2336 and 129Pt from GenBank were used to determine the sizes of the amino acid sequences of the putative ComC and ComE proteins. These sequences were compared with those of H. influenzae strain KW20 and Pasteurella multocida strain Pm70, both of which are closely related to H. somni. The percent identity of ComC and ComE proteins from H. somni to the same proteins of H. influenzae and P. multocida was determined by BLAST from the National Center for Biotechnology Information. The open reading frames (ORFs) of comC and comE from H. somni strain 2336 were determined using SeqBuilder, and the DNA sequences of H. somni comC and comE were aligned with those of H. influenzae using the Clustal-W option of MegAlign, both from DNASTAR Lasergene-10. Genomic DNA from H. influenzae strain KW20 was used as a positive control for all PCR assays. 3. Results 3.1. Natural transformation using genomic or plasmid DNA in strains 2336 and 129Pt Insertion of KanR within the lob2A gene of strain 2336 with 0.2 or 1 mg of genomic DNA from strain 738Dlob2A by natural transformation was successful (Fig. 2), but was of low efficiency. Transformation of strain 2336 with 0.2 mg of DNA resulted in a transformation efficiency of 9.37  1010, whereas transformation of strain 2336 with 1 mg of genomic DNA resulted in a transformation efficiency of 1.2–1.5  109, (results from several transformations). In a typical experiment, over 400 kanamycinresistant colonies would initially be isolated on medium

Fig. 2. Mutagenesis of lob2A in H. somni strain 2336 by natural transformation with donor genomic DNA from strain 738Dlob2A. The KanR gene from Tn5 (in 738Dlob2A) or lob2A from strain 2336 (750 bp amplicon) were amplified by PCR (lanes 1–4 and 6–9, respectively). Lanes and strains: 1 and 2, two separate clones of 2336Dlob2A; 3, 2336; 4, 738Dlob2A; 5, 1-kb DNA ladder; 6 and 7, two separate clones of 2336Dlob2A; 8, 2336; 9, 738Dlob2A. Amplicons for KanR (700 bp) were produced from strains 2336Dlob2A and 738Dlob2A, but not from parent strain 2336. An amplicon of lob2a (750 bp) was produced only from strain 2336. This gel figure is representative of the PCR results obtained from all eight kanamycin-resistant clones identified by colony blotting.

containing kanamycin, but relatively few colonies were determined to contain the KanR gene, as determined by colony blotting with a KanR probe and by PCR. Therefore, 1 mg of genomic DNA was used for subsequent transformation experiments. In contrast, recombinant mutants were not isolated when strain 2336 was transformed with genomic DNA from 2336DluxS, or 2336DuspE. The lob2A gene contains the H. influenzae USS 50 -AAGTGCGGT-30 (Fig. 1), but luxS and uspE lack these uptake sequences. Therefore, the presence of a USS sequence may be necessary for successful allelic exchange in H. somni by natural transformation. Glycerol was used to enhance uptake of plasmid DNA across the inner membrane of 3.2  109 cells of strain 2336 by natural transformation, but was of lower efficiency (1.35  1010). Although concentrations of plasmid from 0.3 to 1 mg were tested, 0.5 mg was found to be optimal. Repeated experiments confirmed the low transformation efficiency using plasmid pNS3K. Natural transformation with H. somni strain 129Pt was unsuccessful with DNA from 2336DluxS, 2336DuspE or 738Dlob2A, and with pNS3K. 3.2. Effect of bacterial growth phase or supplementation with cAMP on natural transformation efficiency The transformation efficiency of strain 2336 was examined when the bacteria were grown to four different growth phases. The bacteria were harvested, incubated with 1.0 mg of genomic DNA, and plated as outlined above on CBA with 80 mg/ml kanamycin. The bacteria still in lag phase (60 Klett units) or early-log phase (80 Klett units) failed to yield any kanamycin-resistant transformants. The bacteria grown to mid-log phase (120 Klett units) had a transformation efficiency of 8.05  109, whereas those

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Fig. 3. Detection of lob2A and luxS in H. somni strains. The genes lob2A and luxS (control) were amplified by PCR (750 bp amplicon) using primers lob2AF and lob2AR (lanes 1–6), and LuxS_Forw and LuxS_Rev (lanes 8–13). Strains and lanes: 1 and 8, M14-622; 2 and 9, 649; 3 and 10, 7735; 4 and 11, 8025; 5 and 12, 2336; 6 and 13, negative control (no DNA); 7, 1-kb DNA ladder.

grown to late-log phase (180 Klett units) were slightly less efficiently transformed (1.48  109). We also evaluated whether transformation efficiency in H. somni could be influenced by the presence of cAMP in the transformation medium. Strains 129Pt and 2336 were grown and incubated in M-IV medium supplemented with or without 2 mM cAMP, as described in Materials and methods. One mg of genomic DNA was then added, the incubation continued for 30 min, and the bacteria cultured onto CBA with 80 mg/ml kanamycin. Strain 129Pt was still unable to be transformed, but the addition of cAMP to the M-IV medium improved the transformation efficiency of strain 2336 from 8.23  109 in the absence of cAMP to 1.41  108 in the presence of cAMP. In addition, individual colonies of strain 2336 grown in the presence of cAMP were almost 50% larger in diameter than colonies from cultures not incubated with cAMP. 3.3. Natural transformation efficiency of other H. somni strains Natural transformation of H. somni strain 2336 following incubation with 2 mM cAMP in M-IV medium was consistently reproducible on multiple attempts, with an efficiency ranging from 1–2.4  108. However, no colonies of H. somni strains 649, 8025, 7735, or M14-622, grown as for strain 2336, were recovered on selective medium following transformation with genomic DNA from 738Dlob2A after several attempts. To confirm that transformation in these strains did not fail due to lack of homologous DNA, PCR was used to amplify lob2A from each strain. Amplification of luxS was used as a control (Fig. 3). The expected size amplicon for lob2A was produced from cells of strains M14-622, 649, 8025, and 2336, but not from strain 7735. Of interest was that luxS was amplified from strains 7735, 8025, and 2336, but not from strains

M14-622 or 649. Failure to amplify lob2A from strain 129Pt has previously been reported (Wu et al., 2000). 3.4. Bioinformatic analysis of the com genes of H. somni A BLAST search was performed comparing the competence genes of H. influenzae to both sequenced genomes of H. somni strains 2336 and 129Pt. All the com genes were represented in H. somni except for comD (Table 3). Only 16 bp separated comC from comE in both strains 2336 and 129Pt. In comparison to the comCDE region of H. influenzae or P. multocida, a region of at least 184 bases was deleted in the region between comC and comE of H. somni. The greatest homology with H. influenzae comD was in H. somni HSM_2018, which was annotated as the inner membrane protein translocase YidC. Comparison of the comE gene of H. influenzae to both H. somni 2336 and 129pt indicated there was 76% query coverage to comE in both strains (Evalues of 5  10103 and 1  10961, respectively). PCR was successful in producing the expected size amplicons from comABCFGJLM of strains 2336 and 129Pt (as well as many other strains tested); PCR did not amplify comD or comE, but the comE forward primer was to a 50 sequence of the gene that was missing in H. somni (data not shown). Both ComC and ComE from strains 2336 and 129Pt were equivalent in size (Table 4). ComC from H. influenzae and P. multocida, when compared to H. somni, was only 8 to 9 amino acids larger. All four bacterial strains produced a ComE protein, but the ComE proteins from H. influenzae and P. multocida were 67 to 68 amino acids larger than the same protein of both H. somni strains. BLAST analysis indicated that the ComC and ComE amino acid sequences shared 98 to 100% identity between H. somni strains 2336 and 129Pt. However, H. somni ComC shared only 31% and 32% identity with H. influenzae and P. multocida ComC, respectively, whereas H. somni ComE shared up to 76%

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Table 3 Genes involved in DNA uptake and transformation in H. somni. Locus tag

Gene

comA comB comC comD comE comF comG comJ comL (DNA uptake lipoprotein) comM rec2 (recombination protein) tfoX (sxy) (regulator of competence-specific genes) crp (catabolite gene activator) pilF2 (involved in uptake, but not binding of DNA [type IV pilus biogenesis/stability protein]) pulJ (involved in DNA binding and uptake –related to type II secretion system) pulG (related to type II secretion system [pseudopilin]) pilA (fimbrial subunit) pilB (pilin/fimbriae biogenesis protein) pilC (type II secretion system protein) pilD (peptidase A24A pre-pilin type IV)

H. somni 129Pt

H. somni 2336

H. influenzae Rd

HS_1109 HS_1110 HS_1111 No* HS_1112 HS_1490 HS_1491 HS_1107 HS_0366 HS_0331 HS_1022 HS_1715 HS_0139 HS_1197

HSM_1070 HSM_1069 HSM_1068 No* HSM_1067 HSM_0511 HSM_0510 HSM_1072 HSM_0636 HSM_0202 HSM_1500 HSM_1867 HSM_0004 HSM_0365 (pilW)

HI0439 HI0438 HI0437 HI0436 HI0435 HI0434 HI0433 HI0441 HI0177 HI1117 HI0061 HI0601 HI0957 HI0366

HS_0278 HS_0264 HS_0250 HS_1430 HS_0457 HS_0458

HSM_0151 HSM_0137 HSM_0123 HSM_0217 HSM_0755 HSM_0756

HI0937 HI0938 HI0299 HI0298 HI0297 HI0296

* blastp indicated that ComD had homology to HSM_2018: inner membrane protein translocase component YidC in H. somni 2336 (max and total score 26.2, Query cover 72%, E value 0.42 and Max identity 13%). Table 4 Genomic analyses of putative ComC, ComD, and ComE proteins of H. somni, H. influenzae, and P. multocida. Bacterial strain

H. somni 2336

H. somni 129Pt

H. influenzae Rd KW20

P. multocida Pm70

Genome accession # Length of ComC protein (amino acids) Length of ComD protein (amino acids) Length of ComE protein (amino acids) Percent identity of H. somni 2336 ComC with ComC of other bacteria Percent identity of H. somni 2336 ComE with ComE of other bacteria

CP000947 165 0 377 NAa NA

CP000436 165 0 377 100 98

L42023 173 137 445 31 76

AE004439 174 120 444 32 71

a

Not applicable.

identity with H. influenzae ComE and up to 71% identity with P. multocida ComE. ORF analyses using SeqBuilder indicated that the ATG start codon of comE was 15 bases downstream of the TAG stop codon of H. somni comC. The Clustal-W comparison of DNA sequences revealed that the H. somni comC sequence did not share a high degree of alignment at the DNA level with H. influenzae comC, but there was substantial alignment between H. somni and H. influenzae comE at the DNA level (Table 4). 4. Discussion Natural transformation is a process by which bacteria directly take up exogenous DNA from their environment through their cell membrane, and incorporate new genes into their genetic code. H. influenzae is able to undergo natural transformation by the induction of competence genes (com operon, dprA, pil operon, rec2, and sxy) (Redfield et al., 2005, 2006; VanWagoner et al., 2004), and through recognition of the USS motif 50 -AAGTGCGGT-30 (Redfield et al., 2006). Using a modification of the protocol from Poje and Redfield (2003), H. somni strain 2336 was successfully transformed repeatedly with genomic DNA that contained

a KanR insertion in the glycosyltransferase gene lob2A (Wu et al., 2000). The presence of the KanR gene was confirmed within lob2A, and although transformation was consistent, transformation efficiency was low. The transformation efficiency of strain 2336 grown to mid-log phase was greater than the transformation efficiency of the same bacteria grown to late-log or early stationary phase, but no recombinants were isolated from cultures in lag phase or grown to early log phase. Supplementation of the transformation medium with cAMP further improved the transformation efficiency. However, multiple natural transformation attempts were unsuccessful with genomic DNA from H. somni mutants containing the KanR gene within luxS and uspE. For transformation to be efficient in H. influenzae, homologs of the type-IV pilus proteins, the ComEC/Rec2 membrane channel, and the recognition of USS are required (Redfield et al., 2006). H. somni contains comCE and rec2, and lob2A contains the same USS as H. influenzae. However, neither luxS nor uspE of H. somni contain any of the USS sequences. The lack of USS in both luxS and uspE may have contributed to their unsuccessful transformation into H. somni. However, although the presence of a USS in a DNA fragment may increase the natural transformation efficiency of that DNA by 100-fold,

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the absence of a USS sequence from a gene or DNA fragment does not prevent its uptake in H. influenzae, so long as there are no competing fragments carrying a USS (Poje and Redfield, 2003). Natural transformation was unsuccessful with strains 129Pt, M14-622, 649, 8025, and 7735, even when using genomic DNA from 738Dlob2A. Unlike pathogenic strain 2336, strain 129Pt is a nonpathogenic commensal of the genital tract. It lacks many of the virulence factors present in strain 2336, including immunoglobulin binding protein (IbpA), lipooligosaccharide phase variation and sialylation, serum resistance, and survival in phagocytic cells (Sandal and Inzana, 2010). In addition, genome sequencing has shown that while the genomes of strains 2336 and 129Pt both experienced many bacteriophage- and transposonmediated events, the genome of strain 129Pt was about 256 kb smaller than that of strain 2336, and many genes responsible for the virulence factors described above were truncated, interrupted, or absent (Siddaramappa et al., 2011). Wu et al. (2000) showed that primers to 415-bp and 218-bp regions of lob2A could not be amplified from strain 129Pt. Furthermore, BLAST analysis showed only 19% homology between lob2A from strains 2336 and 129Pt, indicating lob2A is likely to be truncated or rearranged in strain 129Pt. The lob2A gene could also not be amplified from strain 7735. Therefore, the lack of adequate homology in lob2A in these strains could account for the inability to obtain recombinant strains, and not that these strains are incapable of undergoing natural transformation. However, lob2A was amplified from strains 649, 8025, and M14-622, indicating the absence or rearrangement of lob2A is not likely to be responsible for the lack of natural transformation in these strains. Whether additional competence genes are deficient in these strains was not examined, but warrants further investigation. Of particular interest was that luxS could not be amplified from strains M14-622 or 649. Unpublished data from our lab has shown that a mutation in luxS in strain 2336 severely attenuates this strain. However, strains 649 and M14-622 are highly virulent, and the latter strain was recently isolated from a local herd with bovine respiratory disease. Therefore, further investigation will be required to clarify this discrepancy in virulence factors. Redfield et al. (2006) reported that H. somni strain 129Pt contains defects in comD and comE. The 50 portion of comD is fused to comE, resulting in only 67% of the gene being in frame. Our results confirmed this analysis. However, the same defects are present in comD and comE in strain 2336. PCR did not amplify comD or comE from H. somni, as it did for H. influenzae. However, the H. somni comE gene was likely not amplified because the 50 region of the gene to which the forward primer was made is distinct from that of H. influenzae, but the remainder of the gene was quite similar. The overall function of ComD is not fully known, but ComE is part of the type IV pilus system, and contributes to the formation of the membrane channel protein, which is important for DNA uptake (Chen and Dubnau, 2004). The closest homolog to H. influenzae comD in H. somni is yidC, which encodes for an inner membrane protein translocase. It was not determined if YidC could also carry out the function of ComD. Nevertheless, these

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defects may contribute to the low transformation efficiency in H. somni strain 2336. In H. influenzae, strains vary widely (1000-fold) in the amount of DNA they can take up, and some strains do not develop competence. Mutations in competence genes have been identified in some H. influenzae strains. A frameshift defect in comD that truncated the protein and would be polar on comE was identified in H. influenzae strain R3021, which is poorly transformed. Furthermore, deletion of comM decreased transformation efficiency 300-fold (Maughan and Redfield, 2009). Sequence differences between the donor and recipient DNA can also account for lower transformation efficiency (Poje and Redfield, 2003). In addition to the absence of comD in H. somni, there were a substantial number of non-aligned bases identified in comC between H. somni and H. influenzae, suggesting that a wide range of DNA rearrangements have occurred in this gene. We have previously reported that mobile genetic elements, such a bacteriophages and transposons, are present in the H. somni genome, and that strains 2336 and 129Pt have large regions of rearrangements between them (Siddaramappa et al., 2011). Therefore, H. somni has apparently deviated evolutionarily from other members of the family Pasteurellaceae, with significant DNA rearrangements and deletions occurring within comC, comD, and other genes. These rearrangements apparently include base substitutions and deletion of a region of at least 184 bases between comC and comE. The DNA uptake machinery is unable to transport circular molecules (such as plasmids) through the inner membrane unless circumvented by osmotic shock through the addition of glycerol (Poje and Redfield, 2003). For transformation of pNS3K, the addition of 80% glycerol to the transformation mix promoted osmotic glycerol shock, which facilitated the passage of intact pNS3K into the cytoplasm of H. somni. Nonetheless, the transformation efficiency for pNS3K was also low, which may have been due to a combination of the lack of USS in pNS3K and the missing comD gene. Introduction of the missing comD into H. somni in an efficient, temperature-sensitive shuttle vector incorporating the USS may result in an improved genetic system for manipulating and expressing genes in H. somni. Conflict of interest No conflict of interest is declared. Acknowledgements We thank Jason Inzana and Connor Grimes for excellent and essential technical assistance on this work. This work was funded, in part, by 2007-35204-18338 from the United States Department of Agriculture National Research Initiative Competitive Grants Program to T.J.I. References Briggs, R.E., Tatum, F.M., 2005. Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae. Appl. Environ. Microbiol. 71, 7187–7195.

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