Part:BBa_K5246044

C.Crescentus CB2/CB2A hfsA-hfsB-hfsD Part of polysaccharide export apparatus

Introduction

Caulobacter crescentus is a common freshwater gram-negative oligotrophic bacterium of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, C. crescentus daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure called holdfast, allowing them to adhere to surfaces and perform cell division.[1][2]</p>

Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface[3][4].

The C. crescentus holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in Escherichia coli.

The holdfast synthesis (hfs) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway[4][5][6].

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1740
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1740
  Illegal NotI site found at 475
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1740
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1740
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1740
  Illegal NgoMIV site found at 151
  Illegal NgoMIV site found at 343
  Illegal NgoMIV site found at 352
  Illegal NgoMIV site found at 924
  Illegal NgoMIV site found at 1087
  Illegal NgoMIV site found at 2026
  Illegal NgoMIV site found at 2672
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental characterization

Part cloning

All of the proteins composing this system are responsible for polysaccharide polymerization and export. Since the system's proteins are found in the membrane, we concluded that using a low-copy plasmid would decrease the probability of inclusion body formation. Their formation would diminish the functionality of our system, as the proteins would not allow the polysaccharide to be exported outside the bacteria.

To assemble specifically this part into BBa_K5246046 to then further assemble the holdfast synthesis pathway in E. coli , we had to assemble this part first into a backbone of pACYC-Duet-1 with other composite part genes: BBa_K5246046 . We designed a strategy to maximize the success of plasmid assembly by first assembling plasmids with 3 genes and, after verifying the sequences, integrating 3 left genes into that backbone (Fig. 1). In this way, we prevented Golden Gate assembly errors by trying to construct plasmids from 8 or more fragments.

Fig. 1. Plasmid construction strategy. Plasmids are constructed in two rounds, cloning 3 genes at a time. Verified by colony PCR, restriction digestion analysis, and Nanopore sequencing

The assembly was done using Golden Gate assembly with IIS AarI restriction enzyme sites introduced during PCR amplification. The backbone of pACYC-Duet-1 (Novagen) and fragments were amplified using Phusion Plus DNA polymerase, as the genome of C. crescentus has a high GC% content making the appearance of non-specific products during PCR amplification more common and primer design more challenging (Fig. 2). Since, hfsA gene had an AarI RE site directly in the gene, this site was domesticated during side directed mutagenesis.

Fig. 2 Plasmid construction strategy. Plasmids are constructed in two rounds, cloning 3 genes at a time. Verified by colony PCR, restriction digestion analysis, and Nanopore sequencing

Due to the high amount of non-specific products, the fragments were gel-purified. Vectors and fragments composing this operon, were mixed in equimolar amounts with GG reaction components and incubated as described in protocol. The reaction was later transformed into E. coli Mach1 (Thermo Scientific) competent cells. The assembly was then confirmed with restriction digest analysis (Fig. 3) and positive colonies were sequenced.

Fig. 3 Restriction digest analysis of C. crescentus CB2 pACYC-hfsA-hfsB-hfsD. On the left - expected in silico profile of restriction digest with EcoRI and ScaI, on the right - digested plasmids - 1-6 colonies, M - molecular weight ladder, GeneRuler DNA Ladder Mix (Thermo Scientific)

Full functional project operon assembly with this part and full operon charazterization can be found in composite part BBa_K5246046.